Methods for using transcription-dependent directed evolution of aav capsids

ABSTRACT

Disclosed are methods for performing transcription-dependent directed evolution (TRADE) and novel AAV capsids selected using such methods. This disclosure also provides novel AAV capsid mutants. TRADE technology was used to identify novel AAV vectors that mediate neuronal transduction in the brain following intravenous administration. Application of TRADE in vivo resulted in the identification of new AAV capsids that can transduce neurons more efficiently and more specifically than AAV9 in the brain following administration of the new AAV capsids. The disclosed methods may be used to identify AAV capsids that target various cell populations.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NS088399awarded by the National Institutes of Health/National Institute ofNeurological Disorders and Stroke. The government has certain rights inthe invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001.1] This application is the U.S. National Phase application ofPCT/US2020/016273, filed on Jan. 31, 2020, which claims the benefit ofU.S. Provisional Application No. 62/799,603 filed on Jan. 31, 2019, allof which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0001.2] The instant application contains a Sequence Listing which hasbeen submitted electronically in ASCII format and is hereby incorporatedby reference in its entirety. Said ASCII copy, created on Jan. 21, 2022,is named 60255-701-831-SL.txt and is 108,976 bytes in size.

TECHNICAL FIELD

This disclosure relates to viral vectors used in gene delivery. Morespecifically, this disclosure relates to a method fortranscription-dependent directed evolution and adeno-associated virus(“AAV”) vectors that are selected by using this method.

BACKGROUND

Recombinant adeno-associated virus (“AAV”) vectors are among the mostpromising for in vivo gene delivery. The usefulness of AAV vectors hasbeen expanded since a number of naturally occurring new serotypes andsubtypes were isolated from human and non-human primate tissues. Gao etal., J Virol 78, 6381-6388 (2004) and Gao et al., Proc Natl Acad Sci USA99, 11854-11859 (2002). Among the newly-identified AAV isolates, AAVserotype 8 (AAV8) and AAV serotype 9 (AAV9) have gained attentionbecause AAV vectors derived from these two serotypes can transduce avariety of organs including the liver, heart, skeletal muscles andcentral nervous system with high efficiency following systemicadministration. Ghosh et al., Mol Ther 15, 750-755 (2007); Pacak et al.,Circ Res 99, 3-9 (2006); Inagaki et al., Mol Ther 14, 45-53 (2006); Zhuet al., Circulation 112, 2650-2659 (2005); Wang et al., Nat Biotechnol23, 321-328 (2005); Nakai et al., J Virol 79, 214-224 (2005); and Foustet al., Nature Biotechnol 23, 321-328 (2009). This robust transductionby AAV8 and 9 vectors has been ascribed to strong tropism for these celltypes, efficient cellular uptake of vectors, and/or rapid uncoating ofvirion shells in cells. Thomas et al., J Virol 78, 3110-3122 (2004). Inaddition, emergence of capsid-engineered AAV vectors with betterperformance has significantly broadened the utility of AAV as a vectortoolkit. Asokan et al., Mol Ther 20, 699-708 (2012).

A proof-of-concept using AAV-mediated gene therapy has been shown inmany preclinical animal models of human diseases. Phase I/II clinicalstudies have shown promising results for the treatment for hemophilia B(Nathwani et al., N Engl J Med 71, 1994-2004 (2014)), lipoprotein lipasedeficiency (Carpentier et al., J Clin Endocrinol Metab 97, 1635-1644(2012)), Leber congenital amaurosis (Jacobson et al., Arch Ophthalmol130, 9-24 (2012) and Pierce and Bennett, Cold Spring Harb Perspect Med5, a017285 (2015)), among others (reviewed in Mingozzi and High, Nat RevGenet 12, 341-355 (2011) and Wang et al., Nat Rev Drug Discov 18,358-378 (2019)).

Despite this promise, human studies have also revealed unexpected issuesand potential challenges in AAV-mediated gene therapy. Manno et al., NatMed 12, 342-347 (2006). In addition, despite rapid progress in ourunderstanding of AAV biology and capsid-phenotype relationships (Adachiet al., Nat Commun 5, 3075, (2014); Grimm et al., Hum Gene Ther 28,1075-1086, (2017); and Ogden et al., Science 366, 1139-1143, (2019)),there remain many desirable properties for clinical AAV vectors that wecannot rationally design.

To this end, high throughput screening methods for identifying novel AAVcapsids with such desirable phenotypes have been employed. Inparticular, the development of in vivo AAV library selection strategieshave produced a variety of designer AAV variants capable of highlyefficient transduction of previously refractory cell types (reviewed inKotterman and Schaffer, Nat Rev Genet 15, 445-451 (2014) and Grimm etal., Mol Ther 23, 1819-1831 (2015)).

The earliest attempts at in vivo library selection (1st Generation)relied on recovery of vector genome DNA from dissected tissue.Theoretically, this strategy results in recovery of both effective AAVvariants, as well as AAV variants that mediate some, but not all of thesteps required for vector-mediated transgene expression (FIG. 1 ). Thus,screening a diverse library of synthetic AAV variants potentially leadsto a high background recovery of AAV variants that are completelyineffective gene therapy vectors. Furthermore, targeting a specific celltype requires further processing, such as fluorescence-activated cellsorting or laser capture microdissection. Nonetheless, there have beenseveral reports of successfully employing this technology. Excoffon etal., Proc Natl Acad Sci U S A 106, 3865-3870 (2009); Grimm et al., JVirol 82, 5887-5911 (2008); Lisowski et al., Nature 506, 382-386 (2014);and Dalkara et al., Sci Transl Med 5, 189ra176 (2013). However, alandmark study in 2016 by Deverman et al. showed that this process couldbe greatly improved upon by using a Cre-dependent selection strategy(2nd Generation). Deverman et al., Nat Biotechnol 34, 204-209 (2016).Cre-dependent library selection takes advantage of the selective abilityof Cre recombinase to act on double-stranded DNA, but notsingle-stranded DNA, in order to invert vector genome DNA containing aprimer binding sequence. Inversion of this sequence allows fordirection-selective PCR to specifically amplify viral DNA delivered tocells by AAV variants that are able to undergo the late stage oftransduction at which double stranded DNA is formed from single-strandedAAV genomes. In addition, the use of Cre driver lines facilitatesselective expression of Cre recombinase in a cell type-specific manner,allowing for selection of novel AAV variants that efficiently transduce.Indeed, the use of Cre-dependent selection allowed the authors todevelop an AAV9 variant, AAV-PHP.B, that is capable of 40 times greatertransduction than the parental AAV9 following systemic administration inC57BL/6J mice. Deverman et al., Nat Biotechnol 34, 204-209 (2016).Unfortunately, it has recently become clear that the enhancementexhibited by AAV-PHP.B in mice does not translate to the non-humanprimate context (Matsuzaki et al. 2018 and Hordeaux et al. 2019).Surprisingly, the enhancement does not even extend to all commonly usedmouse strains (Matsuzaki et al. 2018 and Hordeaux et al. 2019). Thereis, therefore, a strong impetus to accelerate the development ofclinically relevant AAV vectors by performing AAV library selectionexperiments in primate models. However, unlike the AAV variant selectionin mice where a plethora of cell type-specific transgenic Cre driverlines are already established, Cre-dependent selection is not tractablein clinically relevant large animals, including non-human primates,because Cre transgenic animals are not readily available.

We therefore sought to develop a next-generation selection strategy (3rdGeneration) with similar or better selective stringency as that providedby Cre-dependent selection, but without the need for Cre recombinase. Inorder to accomplish this goal, we developed the TRAnscription-dependentDirected Evolution system, or TRADE. In the transcription-dependentselection, we express the AAV cap gene as a non-coding antisense mRNAdriven by a cell type-specific enhancer-promoter. Recovery of thisantisense transcript by RT-PCR allows for stringent recovery of AAV capgenes at the level of vector-mediated mRNA expression in a specific celltype without the use of Cre recombinase. Targeting of different celltypes merely requires cloning of a different cell type-specificenhancer-promoter into the plasmid construct. Thus, TRADE is a highlyflexible system that can be applied in a wide variety of contexts,including the non-human primate context for development of enhanced AAVvectors for clinical gene therapy. Note that the same principle can beused for expressing AAV cap gene in an sense orientation. However, thesense strand approach results in expression of immunogenic capsidproteins in target cells and is therefore less ideal than the antisensestrand approach employed by the TRADE system.

SUMMARY

This disclosure provides a next-generation directed evolution strategy,termed TRAnscription-dependent Directed Evolution (“TRADE”), thatselects for AAV capsid transduction at the level of cell type-specificor ubiquitous mRNA expression. The method described herein provides thefollowing advantages over Cre recombination-based AAV targeted evolution(“CREATE”), the most contemporary methods for AAV capsid directedevolution reported in the literature. Deverman et al., Nat Biotech 34,204-209 (2016). First, the CREATE system requires Cre expression, whichcan be attained either by exogenously-delivered Cre expression or by theuse of Cre-transgenic animals. In contrast, the TRADE system does notrequire Cre-transgenic animals; therefore, it can be applied to animalsand cultured cells derived from any animal species and can be readilyadapted to large animals, including non-human primates. Second, unlikethe CREATE system, in which the cell-type specific selection is appliedat the level of AAV viral genome conversion from single-stranded DNA todouble-stranded DNA, TRADE allows for cell type-specific selection atthe level of AAV genome transcription. Therefore, the TRADE system canprovide greater selective pressure than the CREATE system. Third,multiple directed evolution schemes (e.g., neuron-specific,astrocyte-specific, oligodendrocyte-specific, and microglia-specific)can be integrated into one AAV capsid library and selection for AAVvectors targeting each cell type can be performed in a single animal.Fourth, any cell type-specific or tissue/organ-specificenhancers/promoters or ubiquitous enhancers/promoters can be readilyused for AAV capsid directed evolution aimed at identification of celltype-specific or ubiquitous novel AAV capsids with enhanced potency.Fifth, the TRADE methodology is not limited to the genusDependoparvovirus, including the common AAVs that have been used forgene delivery, but can also be applied more broadly to the familyParvoviridae, including in the genera Bocaparvoviruses andErythroparvoviruses other than AAV (e.g., bocaviruses), and even morebroadly to an DNA virus.

This disclosure also provides novel AAV capsid mutants. TRADE technologywas used to identify novel AAV vectors that mediate neuronaltransduction in the brain following intravenous administration.Application of TRADE in C57BL/6J mice and a rhesus macaque resulted inthe identification of new AAV capsids that can transduce neurons moreefficiently and more specifically than AAV9 in the mouse and non-humanprimate brain following intravenous administration. In addition, weidentified a novel AAV capsid that can transduce an undefined cellpopulation or populations, that reside in the lung and are potentiallyof neuronal origin, 5 to 18 times better than the AAV9.

The present disclosure also provides a method to prevent splicing ofantisense mRNA of the AAV capsid gene. Antisense pre-mRNA transcribedfrom the AAV cap gene open reading frame (“ORF”) can be spliced making(a) truncated mRNA species. To our knowledge, this is a new discoverythat has never previously been reported. Such splicing has the potentialto hinder effective recovery of full-length antisense mRNA of the AAVcap ORF, which is essential for TRADE when a wide region of the cap ORFis mutagenized. This disclosure provides a novel strategy to preventsplicing of antisense mRNA of the cap gene.

The TRADE system described herein uses antisense mRNA to recover capsidsequence information, TRADE using sense strand mRNA (i.e., sense strandTRADE) is also feasible using the same principle. However, it should benoted that the sense strand TRADE approach results in expression ofimmunogenic capsid proteins in target cells and therefore is presumablyless ideal than the antisense strand approach.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 An overview of in vivo library selection strategies utilized fordirected evolution of the AAV capsid. AAV vector-mediated transductionis a multi-step process that requires the virion to overcomeextracellular barriers, bind receptors on the target cell, enter thecell via endocytosis, escape the endosome, traffic to the nucleus,uncoat, achieve a double-stranded DNA configuration, and finally undergotranscription/translation. The earliest strategies for in vivo libraryselection (1^(st) Gen) recovered all vector genome DNA from a tissuesample. Theoretically, this strategy would recover both effective AAVvariants, as well as AAV variants that mediate some, but not all of thesteps required for vector-mediated transgene expression. In addition,this strategy would also recover AAV vector genome DNA from AAV vectorparticles that do not enter cells and stay in the extracellular matrix.Thus, screening a diverse library of synthetic AAV variants would leadto a relatively high background recovery of AAV variants that arecompletely ineffective gene therapy vectors. Furthermore, focusing on aspecific cell type requires further processing, such asfluorescence-activated cell sorting (FACS) or laser capturemicrodissection (LCM). The second generation of library selection(2^(nd) Gen) substantially increased selection stringency by utilizingCre-dependent recovery of only those AAV variants that are able toachieve the double-strand DNA stage of transduction. Furthermore,driving the expression of Cre with a cell type-specificenhancer-promoter allows for targeting of a specific cell type whileretaining the benefits of processing bulk tissue samples. The thirdgeneration of library selection (3^(rd) Gen) further builds on AAVdirected evolution technology by employing transcription-dependentrecovery of AAV variants that are able to mediate transgene mRNAexpression from a cell type-specific enhancer-promoter, without therequirement of Cre expression.

FIG. 2 Principle of TRADE. (A) A map of the AAV vector genome in a TRADEconfiguration (AAV-TRADE). A cell type-specific enhancer-promoter isplaced in an antisense orientation to drive AAV cap gene transcriptionexpression as antisense mRNA. A polyadenylation signal (pA) derived fromthe simian virus 40 (SV40) genome is placed within the AAV genome intronin an antisense orientation to terminate antisense AAV cap gene mRNAtranscription. The eGFP open-reading frame (ORF) can be placed asdepicted to serve as a reporter or facilitate enrichment of transducedcells by FACS; however, such a marker gene is not strictly necessary forTRADE. A ubiquitous promoter such as the CAG promoter can be also usedin TRADE in placed of cell type-specific enhancers-promoters to identifyAAV capsids that can transduce a variety of cell types. A celltype-specific enhancer-promoter can be placed upstream of the AAV capgene ORF to drive expression of the AAV cap gene mRNA transcripts in asense orientation (i.e., sense strand TRADE). However, this approach maynot be ideal for TRADE because AAV capsid protein would be expressed intarget cells, which may result in undesired biological consequences inthe directed evolution process. (B) During AAV vector production inHEK293 cells, and in the presence of the adenoviral helper functions,the AAV2 viral p40 promoter drives cap gene expression (forwardtranscription) and cell type-specific transcripts are suppressed,leading to successful production of recombinant AAV vectors containingthe AAV-TRADE vector genome. Following transduction of a specific celltype, the cell type-specific enhancer-promoter is activated, drivingexpression of eGFP and the antisense cap mRNA sequence, while thetranscriptional activity of the p40 promoter remains inactive intransduced cells due to a lack of adenoviral helper functions. Theentire cap gene ORF can be recovered by reverse transcription (RT)-PCRusing antisense cap gene mRNA as a template that is expressed in a celltype-specific manner. We have observed that recombinant AAV vectors canbe produced successfully at high levels even in the presence ofantisense mRNA transcripts expressed due to leaky expression from thehuman synapsin I gene (hSynl) enhancer-promoter in HEK293 cells. We havealso observed that recombinant AAV vectors can be produced successfullyat high titers even when we use the CAG promoter that drives expressionof antisense AAV cap mRNA transcripts at high levels.

FIG. 3 Validation of the TRADE system targeting brain neurons. (A) A mapof the AAV-PHP.B-hSynl-GFP-TRADE vector genome. (B) To verify the TRADEsystem, this AAV vector genome was packaged into the AAV-PHP.B capsid asa single-stranded DNA genome and the resulting AAV vector was injectedinto two 8-week-old C57BL/6J mice intravenously at a dose of 3 × 10¹¹vector genomes (vg) per mouse. Brain tissue was harvested 12 dayspost-injection. The brain tissue from one animal was fixed with 4%paraformaldehyde and used for immunofluorescence microscopy and thebrain tissue from the other animal was unfixed and used for molecularanalysis of AAV vector genome DNA and RNA. (C) Immunofluorescencemicroscopy image of brain sections stained with anti-GFP antibodyconfirmed expression of the cell type-specific enhancer-promoter-driventranscript. (D) hSynl enhancer-promoter-driven GFP expression wasobserved specifically in neurons (anti-HuC/D+). (E) RT-PCR was used torecover the full-length cap ORF sequence (RT+). RT-, a no reversetranscriptase control; Plas, a positive control obtained with DNA-PCRusing a plasmid template containing the AAV-PHP.B-hSynl-GFP-TRADE vectorgenome sequence; NT, a no template PCR control. (F) Sanger sequencing ofthe RT-PCR product revealed expected splicing of the MVM intron in theantisense transcripts expressed by the hSynl enhancer-promoter (SEQ IDNO:190). The exon-exon junction is highlighted with gray. (G) Sangersequencing confirmed the insertion of the PHP.B peptide (highlightedwith gray) (SEQ ID NO:191).

FIG. 4 Splicing of the antisense mRNA of the AAV9 cap ORF. Two celllines, HEK293 and Neuro2a, were transfected with plasmids containing theAAV9 cap ORF in the TRADE configuration, with or without a GFP reporter.They are indicated as “GFP TRADE” and “TRADE”, respectively, in thefigure. Cells were harvested 3 days post-transfection, RNA wasextracted, and RT-PCR was performed with a set of PCR primers thatamplify the full cap ORF sequence. Instead of recovering the expectedamplicon size of 2.4 kb as shown in the positive control (PC) lane, weconsistently recovered amplicons of approximately 0.7 kb. Sangersequencing of these RT-PCR products identified a truncation consistentwith splicing of a 1.7 kb region of the AAV9 cap ORF indicated in FIG. 5. PC, a positive control using a plasmid template containing theAAV-PHP.B-hSynl-GFP-TRADE vector genome sequence; NC, a no template PCRcontrol.

FIG. 5 An intron identified in antisense mRNA derived from the AAV9 capgene (SEQ ID NO:192). When the AAV-PHP.B cap gene sequence wastranscribed in an antisense orientation in HEK293 cells or Neuro2a cellsunder the control of the neuron-specific human synapsin I (hSynl)enhancer-promoter, a splicing event was identified with cryptic splicedonor and splice acceptor sites (please refer to FIG. 6 as well). Theunderlined sequence indicates the intron found within the AAV9 cap ORF.This splicing event was not observed in mouse brain neurons. It shouldbe noted that (1) although the hSynl enhancer-promoter has been used asa neuron-specific element, it has been shown to drive leaky expressionin HEK293 cells; and (2) the AAV9 cap ORF sequence used for the intronsplicing experiment had the following silent mutations near theC-terminus: gaaccccgccccattggcacGCgTtacCTGACTCGTAATCTGTAA (SEQ ID NO:1).The intron sequence is underlined, and the silent mutations that havebeen introduced into the intron to create an Mlul (ACGCGT) recognitionsite are indicated in uppercase.

FIG. 6 Cryptic splice donor (SD) and splice acceptor (SA) sites with thecommon features of exon-intron junctions present in the AAV cap ORFs inan antisense orientation. Nucleotide sequences of the cap genes derivedfrom 122 naturally occurring AAV strains (serotypes and variants) arealigned using a multiple sequence alignment program (SEQ ID NO 223-316).The exon-intron junctions identified in the AAV9 cap ORF-derivedantisense mRNA are indicated with solid lines. The dashed line in thesplice acceptor region indicates putative splice acceptor sites in theAAV cap ORFs devoid of the splice acceptor AG/TC sequence at theposition expected from the sequence conservation. The dashed line in thesplice donor region indicates the splice donor site identified in theAAV3 cap ORF-derived antisense mRNA (please refer to FIG. 7 ). The GT/CAsplice donor sites and the AG/TC splice acceptor motifs, followed by astretch of T’s, are the common features of exon-intron junctions and arevery well-conserved across many AAV strains. The splice donor andacceptor sites identified in the AAV9 cap ORF shown in this figure havealso been identified in the AAV1 cap ORF. For serotypes other than AAV1,3, 5 and 9, splicing events in antisense mRNA of the AAV cap ORFs arecurrently under investigation. The highlighted variants are common AAVserotypes.

FIG. 7 Introns identified in antisense mRNA derived from the AAV3 capgene. pAAV3-hnLSP-MCS-TRADE2 is a plasmid carrying the wild-type AAV3cap ORF placed under a liver-specific enhancer-promoter with an MVMintron (hnLSP). The nucleotide sequence of the AAV3 cap ORF is the sameas that of the naturally identified AAV3. HepG2 cells, a human hepatomacell line, were transfected with plasmid pAAV3-hnLSP-MCS-TRADE2.Antisense mRNA derived from the AAV3 cap ORF was then analyzed byRT-PCR. Sequences of two truncated RT-PCR products were determined bySanger sequencing following blunt-end TOPO cloning of the PCR products,which revealed introns found within the antisense AAV3 cap ORF (Panels Aand B, SEQ ID NO:193). Intron sequences are in lowercase letters withunderline. The most upstream splice donor site is found to be only 3 bpaway from the splice donor site identified in the AAV9 cap ORF, which isindicated in a dashed line in FIG. 6 . The most downstream spliceacceptor site is found approximately 80 bp upstream of that of the AAV9cap ORF. Please note that all the splice donor and acceptor sitesidentified in the AAV3 cap ORF have also been identified in the AAV1 capORF.

FIG. 8 . Additional cryptic splice acceptor sites present in the AAV capORFs. (A and B) Nucleotide sequences of the cap genes derived from 122naturally occurring AAV strains (serotypes and variants) are alignedusing a multiple sequence alignment program (SEQ ID NO:317-420). Theexon-intron junctions at the splice acceptor sites identified in theAAV3 cap ORF-derived antisense mRNA are indicated with solid thin lines.The dashed line in Panel A indicates alternative putative spliceacceptor sites near the experimentally determined splice acceptor site.The AG/TC splice acceptor sites, followed by a stretch of T’s, are acommon feature of exon-intron junctions at splice acceptor sites and arevery well conserved across many AAV strains. The AAV3 cap ORF ishighlighted. The splice acceptor sites identified in the AAV3 cap ORFshown in Panels A and B have also been identified in the AAV1 cap ORF.As for the AAV5 cap ORF, no splicing events have been observed at anysites in antisense mRNA transcription. For serotypes other than AAV1, 3,5 and 9, splicing events in antisense mRNA of the AAV cap ORFs arecurrently under investigation.

FIG. 9 Additional potential splice donor sites present in the AAV capORFs. Nucleotide sequences of the cap genes derived from 122 naturallyoccurring AAV strains (serotypes and variants) are aligned using amultiple sequence alignment program (SEQ ID NO:421-461). The exon-intronjunctions at the splice donor sites identified in the AAV3 capORF-derived antisense mRNA are indicated with a solid line. The GT/CAsplice donor consensus sequence at this position is retained by onlyhalf of AAV strains. This splice donor site has been identified in theAAV1 cap ORF.

FIG. 10 Splice donor and splice acceptor sites identified in the AAV1cap ORF. The nucleotide sequence of the AAV1 cap ORF is shown (SEQ IDNO:194). The AAV1 cap ORF was expressed by the hSynl enhancer-promoterin human embryonic kidney (HEK) 293 cells or Neuro2a cells in anantisense orientation. Antisense mRNA derived from the AAV1 cap ORF wasthen analyzed by RT-PCR. Sequences of RT-PCR products were determined bySanger sequencing following blunt-end TOPO cloning of the PCR products,which revealed introns found within the AAV1 cap ORF. Exon-intronjunctions identified in antisense AAV1 cap mRNA are indicated with AG/TCfor the splice donor sites and GT/CA for the splice acceptor sites.AG/TC and GT/CA in uppercase are the consensus two nucleotides at the 5′end and the 3′ end of an intron, respectively. Since the splicing occursin antisense mRNA of the ORF, intron sequences are between CT (spliceacceptor) and AC (splice donor) in various combinations in the abovesequence. The detailed information about the observed combinations ofthe splice donors and acceptors is not shown. The two conservednucleotides at exon-intron junctions (CT or AC) indicated in boldfaceare those that are highly conserved across different AAV serotypes. Thetwo conserved nucleotides at exon-intron junctions (CT or AC) that areunderlined are those that have also been identified in antisense AAV3 orAAV9 cap mRNA transcripts.

FIG. 11 Splicing-suppressing mutagenesis of the AAV9 cap ORF. Silentmutations are introduced around the splice acceptor (SA) site and/or thesplice donor (SD) site in the AAV9 cap ORF to suppress the splicingobserved on the antisense mRNA transcripts. The spliced-out intron fromthe native sequence (SEQ ID NO:195, SEQ ID NO:196) is indicated withunderlines. The AAV9NS1 genome (SEQ ID NO:197) has a set of mutationsaround the SA site while the AAV9NS2 genome (SEQ ID NO:198) has a set ofmutations around the SD site. The AAV9NS3 genome has both sets ofmutations. The numbers to the right indicate the nucleotide positionrelative to the first nucleotide of the AAV9 cap ORF.

FIG. 12 Mutations introduced around the splice donor and/or acceptersite(s) effectively suppress the splicing of antisense mRNA derived fromthe AAV9 cap ORF. Neuro2a cells were transfected with plasmidscontaining the AAV9 cap ORF and various potentially splicing-suppressingmutations in the TRADE configuration (NS1-3). RNA was harvested 3 dayspost-transfection and RT-PCR was performed with a set of PCR primersthat can recover the full cap ORF sequence. In stark contrast to resultsseen in FIG. 4 , full-length amplicons were successfully recovered. NS1,the AAV9-TRADE vector genome with a codon-modified splice acceptor. NS2,the AAV9-TRADE vector genome with a codon-modified splice donor. NS3,the AAV9-TRADE vector genome with codon-modified splice acceptor andsplice-donor. PC, a positive control using a plasmid template containingthe AAV-PHP.B-hSynl-GFP-TRADE vector genome sequence; NC, a no templatePCR control.

FIG. 13 Study design for application of TRADE to identify enhanced AAVvariants for brain neuron transduction following systemic AAV vectorinjection. (A) A map of the AAV9-N272A-hSynl-GFP-TRADE-PepLib vectorgenome. The hSynl enhancer-promoter is utilized to drive expressionspecifically in neurons. The liver-detargeted AAV9-N272A cap(PCT/US2017/068050) serves as the platform for AAV library generation. Arandomized 8 amino acid peptide encoded by (NNK)₈ and flanked byglycine-serine linkers (SEQ ID NO:2) was substituted for Q588 of theAAV9-N272A cap sequence (SEQ ID NO:222). (B) The plasmid library wasused to produce an AAV library using a triple transfection protocol. Thelibrary was purified through PEG precipitation and two rounds of CsCIultracentrifugation, then injected via tail vein at a dose of 3 x 10¹¹vg/mouse. Brain tissue was harvested 12 days post-injection. RNA wasrecovered using TRIzol and RT-PCR was used to recover a fragment of capcontaining the peptide insertion, which was subsequently cloned backinto the AAV vector plasmid backbone. This was repeated for 3 rounds ofselection in C57BL/6J mice. In parallel, a single round of selection wasperformed in rhesus macaque using a dose of 2.7 x 10¹² vg/kg.

FIG. 14 Validation of neuronal transduction of the 26 novel AAV capsidsin mice and a nonhuman primate by AAV RNA Barcode-Seq. (A) A map of thedouble-stranded (ds) AAV-hSynl-GFP-BC vector. A pair of two 12nucleotide-long DNA barcodes (VBCx-L and VBCx-R) are placed under thehuman synapsin I (hSynl) gene enhancer-promoter. These two virusbarcodes (VBCs) can be expressed as transcripts specifically in cellswhere the hSynl enhancer-promoter is active (i.e., neurons). (B)Neuronal transduction of 26 novel AAV variants, HN1 to HN26, identifiedby TRADE (5 variants identified in mice and 21 variants identified in anon-human primate) and 3 control AAV capsids (AAV9, AAV9-N272A andAAV-PHP.B) in C57BL/6J and BALB/cJ mice. A DNA/RNA-barcodeddsAAV-hSynl-GFP-BC library (dsAAV-hSynl-GFP-BCLib) containing 26 novelAAV variants identified by TRADE (5 variants identified by TRADE in miceand 21 variants identified by TRADE in a non-human primate) and controlAAV capsids (AAV9, AAV9-N272A and AAV-PHP.B) was injected intravenouslyinto three adult male C57BL/6J mice and three adult male BALB/cJ mice ata dose of 5 x 10¹¹ vg per mouse. Two weeks post-injection, varioustissues were harvested and analyzed for transduction at AAV vectorgenome transcripts levels by AAV RNA Barcode-Seq. Transduction levelsare expressed as phenotypic difference (PD) values relative to thereference control, AAV9. For the AAV capsid amino acid sequenceinformation of the HN1 to HN26 variants, please refer to Table 3. (C)Neuronal transduction of the 26 novel AAV variants and 3 control AAVcapsids in the hippocampus of a rhesus macaque. The sameDNA/RNA-barcoded AAV library was injected intravenously into onejuvenile male rhesus macaque at a dose of 2 × 10¹³ vg/kg. Two weekspost-injection, various brain regions were harvested and analyzed fortransduction by AAV RNA Barcode-Seq. (D) Relative neuronal transductionefficiencies of 3 TRADE variants, HN1, HN2 and HN3, and AAV-PHP.B wereanalyzed by AAV RNA Barcode-Seq in 12 different brain regions in thesingle rhesus macaque used for Panel C. In Panels B, C and D, dashedlines indicate the PD value of AAV9 (i.e., 1.0).

FIG. 15 Validation of enhanced neuronal transduction of AAV9-N272A-HN1in mice using conventional eGFP reporter vectors and histologicalquantification. We produced AAV9, AAV-PHP.B, and AAV9-N272A-HN1 vectorscontaining self-complementary AAV genomes expressing eGFP under thecontrol of the hSynl enhancer-promoter (dsAAV-hSynl-eGFP). Purifiedvectors were administered via the tail vein at a dose of 3 x 10¹¹vg/mouse into 8-week old male C57BL/6J or BALB/cJ mice (n = 4 mice /vector / mouse strain). Three weeks post-injection, mice weretranscardially perfused with 4% paraformaldehyde and brain tissue wasprocessed for immunohistochemistry. (A) A map of the self-complementaryhSynl-eGFP vector genome. (B) Representative tilescan images of sagittalsections stained with anti-GFP antibody. (C) Quantification of neuronaltransduction in (B) based on automated counts of cells expressing eGFPand NeuN in four brain regions. (D) Validation of the automated countingprocess in (B) and (C). Representative 20X confocal images from visualcortex are shown. Scale bar = 100 µm. (E) Quantification of neuronaltransduction in (D) based on hand counts of cells expressing eGFP andNeuN by a blinded observer. Error bars represent mean +/- SEM.***p<0.001.

FIG. 16 Validation of enhanced AAV9-N272A-HN1 transduction relative toAAV9 in rhesus macaques using epitope-tagged eGFP reporter vectors. (A)AAV-CAG-nlsGFP vectors used for this study. We produced 4 AAV vectors:AAV9-CAG-FLAGnlsGFP-BCLib, AAV9-CAG-HAnlsGFP-BCLib,AAV9-N272A-HN1-CAG-FLAGnlsGFP-BCLib andAAV9-N272A-HN1-CAG-HAnlsGFP-BCLib. The nlsGFP (eGFP with the nuclearlocalization signal derived from the SV40 large T antigen) was taggedwith either the FLAG tag or the HA tag at the N-terminus. Each vectorwas a DNA/RNA-barcoded library containing an approximately 1 to 1mixture of 9 different DNA/RNA-barcoded viral clones; however, thisfeature was not used in this study. The two vectors in the top halfdepicted in Panel A were mixed at a ratio of 1:1 to make AAV Library 1(AAVLib1) and the two vectors in the bottom half were mixed at a ratioof 1:1 to make AAV Library 2 (AAVLib2). In this experimental scheme,AAVLib1 and AAVLib2 each contain AAV9 and AAV9-N272A-HN1 vectorsexpressing epitope-tagged nlsGFP at a ratio of 1:1, but thecapsid-epitope relationship is inverted in order to avoid potentialantibody bias in downstream analyses. (B) Representative tile-scannedbrain section from one animal receiving AAVLib. Each AAV library wasadministered intravenously into a juvenile rhesus macaque at a dose of 3x 10¹³ vg/kg. Tissue was harvested 3-weeks post-injection, cut into 4 mmslabs, fixed in 4% paraformaldehyde, and processed forimmunohistochemical analysis with anti-GFP, anti-FLAG and anti-HAantibodies. eGFP expression indicates that a cell was transduced byeither AAV9 or AAV9-N272A-HN1 or both. FLAG staining indicates that theAAV9 capsid mediated transduction, while HA staining indicates thatAAV9-N272A-HN1 mediated transduction. Top-right inset, motor cortex;bottom-right inset, putamen. This experiment revealed thatAAV9-N272A-HN1 transduced the brain cells better than AAV9 by severalfold with strong neuronal tropism compared to AAV9. Therefore, as far asneuronal transduction is concerned, AAV9-N272A-HN1 mediates much higherneuronal transduction than AAV9.

FIG. 17 Biodistribution of AAV9-N272A-HN1 to major peripheral organsfollowing systemic delivery in mice and rhesus macaques. We used AAV DNABarcode-Seq to determine relative abundance of AAV vector genome DNAs ineach peripheral organ, delivered by each AAV capsid contained in thedsAAV-hSynl-GFP-BCLib library (Panels A, B and C). As explained earlier,the dsAAV-hSynl-GFP-BCLib library contained 26 AAV variants identifiedby TRADE in mice and in a non-human primate together with the controls,AAV9, AAV9-N272A and AAV-PHP.B. DNA was extracted from various tissuesfollowing administration of the dsAAV-hSynl-GFP-BCLib library (see Table3) and subjected to AAV DNA Barcode-Seq analysis. We also used AAV RNABarcode-Seq to determine relative transduction efficiency compared toAAV9 in each peripheral organ of rhesus macaques intravenously injectedwith the ssAAV-CAG-nlsGFP-BCLib library depicted in FIG. 16A (Panel D).(A) Biodistribution of AAV9, AAV9-N272A, AAV-PHP.B, and TRADE variantsto the liver, relative to AAV9, in C57BL/6J mice, BALB/cJ mice andrhesus macaques. (B) Biodistribution of AAV9-N272A-HN1 to majorperipheral organs besides the liver in C57BL/6J mice and BALB/cJ mice (n= 3 mice / strain). (C) Biodistribution of AAV9-N272A-HN1 to majorperipheral organs besides the liver in a rhesus macaque (n = 1) based ondsAAV-hSynl-GFP-BC analysis. For this experiment, AAV DNA Barcode-Seqanalysis was performed on the samples collected from one rhesus macaqueinjected with the dsAAV-hSynl-GFP-BCLib library shown in FIG. 14D. (D)Biodistribution of AAV9-N272A-HN1 to major peripheral organs besides theliver in rhesus (n = 2) based on ssAAV-CAG-nlsGFP-BC analysis. For thisexperiment, AAV RNA Barcode-Seq analysis was performed on the samplescollected from rhesus macaques injected with the ssAAV-GAG-nlsGFP-BCLibvectors shown in FIG. 16A. Error bars represent mean +/- SEM.AAV9-N272A-HN1 capsid transduced peripheral organs to a lesser degreecompared to AAV9 capsid.

FIG. 18 AAV9-N272A-HN1 is highly neurotropic following systemicadministration in mice. AAV9 and AAV9-N272A-HN1 vectors expressingnlsGFP under the control of the strong, ubiquitous CAG promoter wereinjected intravenously into 8-week old male BALB/cJ mice at a dose of 3x 10¹¹ vg/mouse. Tissues were harvested 12 days post-injection andanalyzed by immunostaining with anti-GFP and anti-NeuN antibodies. (A) Amap of the single-stranded (ss) AAV-CAG-nlsGFP vector genomes used inthis study. (B) Representative image from mouse cerebral cortextransduced with AAV9-N272A-HN1-CAG-nlsGFP. The vast majority of cellstransduced with AAV9-N272A-HN1-CAG-nlsGFP are also positive for theneuronal marker NeuN. Scale bar= 100 µm. (C) Neuronal specificity ofAAV9 and AAV9-N272A-HN1 capsids. Quantification of neuronal specificitywas determined by dividing the number of double-positive cells(eGFP+/NeuN+) by the total number of GFP+ cells. AAV9-N272A-HN1 ishighly specific to neurons (96%) compared to AAV9 (56%).

DETAILED DESCRIPTION

In some embodiments, the present disclosure provides a TRADE system thatallows directed evolution of the AAV capsid using antisense mRNA of thecap ORF expressed in a cell type-specific or ubiquitous manner. Such asystem does not require Cre-transgenic animals. Therefore, it can beapplied to cell type-specific AAV capsid evolution in large animals,including non-human primates, for which Cre-transgenic strains are notreadily available. Any cell type-specific or tissue/organ-specificenhancers/promoters or ubiquitous enhancers/promoters can be readilyapplied to the system with no requirement of transgenesis. The celltype-specific selection is given at the mRNA level. In certainembodiments, multiple directed evolution schemes may be combined intoone directed evolution scheme. For example, selection of neuron-specificAAV capsids, astrocyte-specific AAV capsids, oligodendrocyte-specificAAV capsids and microglia-specific AAV capsids based on celltype-specific transgene mRNA expression can be performed simultaneouslyin a single animal.

In some embodiments, the present disclosure provides a sense strandTRADE system that allows directed evolution of the AAV capsid using mRNAof the cap ORF expressed in a cell type-specific or ubiquitous mannerthat is capable of expressing AAV capsid proteins in target cells. Thesense strand TRADE has the same advantages of those antisense strandTRADE presented with data here in that it does not requireCre-transgenic animals, cell type-specific selection is given at themRNA level, and it is capable of combining multiple directed evolutionschemes into one directed evolution round done in a single animal.However, the possible disadvantage is that immunogenic AAV capsidproteins may be unavoidably expressed persistently in target cells,which may result in undesired consequences in the capsid selectionprocess.

In some embodiments, the present disclosure also provides novel AAVcapsids. In certain embodiments, these novel AAV capsids can transducebrain neurons several times better than AAV9 in C57BL/6J mice followingintravenous injection. In certain embodiments, the novel AAV capsidstransduced up to 8 times better than AAV9 in C57BL/6J mice followingintravenous injection. The neuronal transduction levels may be greatlyenhanced compared to AAV9 although they may not attain the levelsobtained with AAV PHP.B. In certain embodiments, the novel AAV capsidsmay transduce brain neurons more efficiently than AAV PHP.B.

In some embodiments, this disclosure provides novel AAV capsids that cantransduce brain neurons several times better than AAV9 followingintravenous injection in BALB/cJ mice. In certain embodiments, the novelAAV capsids can transduce brain neurons up to 7 times better than AAV9following intravenous injection in BALB/cJ mice. The transduction levelsare much higher than AAV PHP.B.

In some embodiments, this disclosure provides novel AAV capsids that cantransduce brain neurons several times better than AAV9 in rhesusmacaques following intravenous injection. In certain embodiments, thenovel AAV capsids can transduce brain neurons up to 4 times better thanAAV9 in rhesus macaques following intravenous injection. Thesetransduction levels are better than AAV PHP.B.

In some embodiments, the disclosure provides AAV capsids that cantransduce the pulmonary cells with neuronal cell marker expressionseveral times better than AAV9. In certain embodiments, the AAV capsidscan transduce such cells up to 17 times better than AAV9.

In some embodiments, the novel AAV capsids exhibit a liver-detargetingphenotype.

In some embodiments, the disclosure provides codon-modified AAV capsequences that are not spliced when expressed in an antisense direction.We have observed that unmodified AAV cap ORFs are spliced when expressedin an antisense direction (e.g., AAV1, AAV3 and AAV9). In contrast, someof the codon-modified AAV cap ORFs described in this disclosure are notspliced. Based on the knowledge we have developed about the putativesplice donor and acceptor sites, it has become possible to design suchnon-spliced versions of AAV cap ORFs. The use of such non-spliced capORFs may be used for directed evolution using the TRADE system whenmutagenesis of the cap gene takes place over a wide region of the capORF.

The term “AAV vector” as used herein means any vector that comprises orderives from components of AAV and is suitable to infect mammaliancells, including human cells, of any of a number of tissue types, suchas brain, heart, lung, skeletal muscle, liver, kidney, spleen, orpancreas, whether in vitro or in vivo. The term “AAV vector” may be usedto refer to an AAV type viral particle (or virion) comprising at least anucleic acid molecule encoding a protein of interest.

Additionally, the AAVs disclosed herein may be derived from variousserotypes, including combinations of serotypes (e.g., “pseudotyped” AAV)or from various genomes (e.g., single-stranded or self-complementary).In particular embodiments, the AAV vectors disclosed herein may comprisedesired proteins or protein variants. A “variant” as used herein refersto an amino acid sequence that is altered by one or more amino acids.The variant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties, e.g., replacement ofleucine with isoleucine. Alternatively, a variant may have“nonconservative” changes, e.g., replacement of a glycine with atryptophan. Analogous minor variations may also include amino aciddeletions or insertions, or both.

Nucleotide sequences, such as polynucleotides, encoding proteins of thepresent disclosure are provided herein. The nucleotides of the presentdisclosure can be composed of either RNA or DNA. The disclosure alsoencompasses those polynucleotides that are complementary in sequence tothe polynucleotides disclosed herein.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode the proteins of the presentdisclosure. In addition, it is well within the skill of a person trainedin the art to create alternative polynucleotide sequences encoding thesame, or essentially the same, proteins disclosed herein. These variantor alternative polynucleotide sequences are within the scope of thecurrent disclosure. As used herein, references to “essentially the samesequence” refers to one or more sequences that encode amino acidsubstitutions, deletions, additions, or insertions that do not eliminatethe detectability of the polypeptide encoded by the polynucleotides ofthe present disclosure.

The current disclosure also includes variants of the polynucleotides andpolypeptides disclosed herein. Variant sequences include those sequenceswherein one or more peptides or nucleotides of the sequence have beensubstituted, deleted, and/or inserted.

Polynucleotide and polypeptide sequences of the current disclosure canalso be defined in terms of particular identity and/or similarity withcertain polynucleotides and polypeptides described herein. The sequenceidentity will typically be greater than 60%, preferably greater than75%, more preferably greater than 80%, even more preferably greater than90%, and can be greater than 95%. The identity and/or similarity of asequence can be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalas compared to a sequence disclosed herein. Unless otherwise specified,as used herein percent sequence identity and/or similarity of twosequences can be determined using the algorithm of Karlin and Altschul(1990), modified as in Karlin and Altschul (1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990). BLAST searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain sequences with the desired percentsequence identity. To obtain gapped alignments for comparison purposes,Gapped BLAST can be used as described in Altschul et al. (1997). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (NBLAST and XBLAST) can be used.

Methods of producing AAV vectors as disclosed herein are well known inthe art, including methods, for example, of using packaging cells,auxiliary viruses or plasmids, and/or baculovirus systems. See, e.g.,Samulski et al., J. Virology 63, 3822 (1989); Xiao et al., J. Virology72, 2224 (1998); Inoue et al., J. Virology 72, 7024 (1998);WO1998/022607; and WO2005/072364.

Methods of producing pseudotyped AAV vectors are also known (see, e.g.,WO00/28004), as well as various modifications or formulations of AAVvectors, to reduce their immunogenicity upon in vivo administration(see, e.g., WO01/23001; WO00/73316; WO04/112727; WO05/005610; andWO99/06562). In some embodiments, AAV vectors may be prepared or derivedfrom various serotypes of AAVs which may be mixed together or mixed withother types of viruses to produce chimeric (e.g., pseudotyped) AAVviruses.

In particular embodiments, the AAV vector may be a human serotype AAVvector. In such embodiments, a human AAV may be derived from any knownserotype, e.g., from any one of serotypes 1-11, for instance from AAV1,AAV2, AAV4, AAV6, or AAV9.

The AAV vectors disclosed herein may include a nucleic acid encoding aprotein of interest. In various embodiments, the nucleic acid also mayinclude one or more regulatory sequences allowing expression and, insome embodiments, secretion of the protein of interest, such as e.g., apromoter, enhancer, polyadenylation signal, an internal ribosome entrysite (“IRES”), a sequence encoding a protein transduction domain(“PTD”), a 2A peptide, and the like. Thus, in some embodiments, thenucleic acid may comprise a promoter region operably linked to thecoding sequence to cause or improve expression of the protein ofinterest in infected cells. Such a promoter may be ubiquitous, cell- ortissue-specific, strong, weak, regulated, chimeric, etc., for example,to allow efficient and stable production of the protein in the infectedtissue. The promoter may be homologous to the encoded protein, orheterologous, although generally promoters of use in the disclosedmethods are functional in human cells. Examples of regulated promotersinclude, without limitation, Tet on/off element-containing promoters,rapamycin-inducible promoters, tamoxifen-inducible promoters, andmetallothionein promoters. Other promoters that may be used includepromoters that are tissue specific for tissues such as kidney, spleen,and pancreas. Examples of ubiquitous promoters include viral promoters,particularly the CMV promoter, the RSV promoter, the SV40 promoter,etc., and cellular promoters such as the phosphoglycerate kinase (PGK)promoter and the β-actin promoter.

In some embodiments of the AAV vectors disclosed herein, one or morefeedback elements may be used to dampen over-expression of the proteinof interest. For example, some embodiments of the AAV vectors mayinclude one or more siRNA sequences that would target the exogenoustranscript. In other embodiments, the AAV vector may include one or moreadditional promoters that may be recognized by inhibitory transcriptionfactors. In various embodiments, the AAV vectors disclosed herein maycomprise a construct that may create a homoeostatic feedback loop thatmay maintain expression levels of the protein of interest at aphysiological level.

In some embodiments of the AAV vectors disclosed herein, genome editingmachinery may be used to genetically modify cellular genome DNA or mRNAtranscripts at a site-specific manner. Komor et al., Cell 168, 20-36(2017); and Katrekar et al., Nature Methods 16:239-242, 2019. Forexample, some embodiments of the AAV vectors may include aCRISPR-associated enzyme such as Cas9, a DNA base editor, an RNA editaseand/or guide RNA (gRNA) to modify nucleic acid in cells in asite-specific manner. In addition, AAV vectors may contain a homologyrepair template (HDR) for genome editing.

In various embodiments, the AAV vectors disclosed herein can comprise anucleic acid that may include a leader sequence allowing secretion ofthe encoded protein. In some embodiments, fusion of the transgene ofinterest with a sequence encoding a secretion signal peptide (usuallylocated at the N-terminal of secreted polypeptides) may allow theproduction of the therapeutic protein in a form that can be secretedfrom the transduced cell. Examples of such signal peptides include thealbumin, the β-glucuronidase, the alkaline protease or the fibronectinsecretory signal peptides.

As described herein, effective and long-term expression of therapeuticproteins of interest in brain, heart, lung, skeletal muscle, kidney,spleen, or pancreas can be achieved with non-invasive techniques,through peripheral administration of certain AAV vectors, such as anon-AAV9 vector with AAV9 sequences. Such peripheral administration mayinclude any administration route that does not necessitate directinjection into brain, heart, lung, skeletal muscle, kidney, spleen, orpancreas. More particularly, peripheral administration may includesystemic injections, such as intramuscular, intravascular (such asintravenous,) intraperitoneal, intra-arterial, or subcutaneousinjections. In some embodiments, peripheral administration also mayinclude oral administration (see, e.g., WO96/40954), delivery usingimplants, (see, e.g., WO01/91803), or administration by instillationthrough the respiratory system, e.g., using sprays, aerosols or anyother appropriate formulations.

In various embodiments, the desired doses of the AAV vectors may beadapted by the skilled artisan, e.g., depending on the diseasecondition, the subject, the treatment schedule, etc. In someembodiments, from 10⁵ to 10¹² viral genomes are administered per dose,for example, from 10⁶ to 10¹¹, from 10⁷ to 10¹¹, or from 10⁸ to 10¹¹. Inother embodiments, exemplary doses for achieving therapeutic effects mayinclude virus titers of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ or10¹¹ viral genomes or more. Virus titer may also be expressed in termsof transducing units, which may be readily calculated by those of skillin the art.

In various embodiments, the AAV vectors disclosed herein may beadministered in any suitable form, for instance, either as a liquidsolution or suspension, as a solid form suitable for solution orsuspension in liquid prior to injection, as a gel or as an emulsion. Thevectors may be formulated with any appropriate and pharmaceuticallyacceptable excipient, carrier, adjuvant, diluent, etc. For instance, forinjection, a suitable carrier or diluent may be an isotonic solution, abuffer, sterile and pyrogen-free water, or, for instance, a sterile andpyrogen-free phosphate-buffered saline solution. For inhalation, thecarrier may be in particulate form.

The vectors may be administered in a “therapeutically-effective” amount,e.g., an amount that is sufficient to alleviate (e.g., decrease, reduce)at least one of the symptoms associated with a disease state, or toprovide improvement in the condition of the subject. In someembodiments, repeated administrations may be performed, for instanceusing the same or a different peripheral administration route and/or thesame vector or a distinct vector.

EXAMPLES

The following examples are for illustration only. In light of thisdisclosure, those of skill in the art will recognize that variations ofthese examples and other embodiments of the disclosed subject matter areenabled without undue experimentation.

We applied the TRADE system in both C57BL/6J mice and a rhesus macaquein order to identify novel AAV capsids that efficiently transduce brainneurons following systemic delivery. The TRADE system utilizes a plasmidconstruct containing an overlapping bicistronic AAV genome flanked byITR sequences (FIG. 2A). In the sense direction, the AAV2 p40 promoterdrives expression of the AAV cap gene to facilitate efficient productionof viral particles (FIG. 2B). In the antisense direction, a celltype-specific enhancer-promoter (e.g. the human synapsin I (hSynl)enhancer-promoter) drives expression of transcripts encoding GFP and theantisense cap sequence (FIG. 2B), terminating at a polyadenylationsignal (poly A) embedded in the intron present in the AAV2 genome.Utilizing the TRADE construct as a cloning backbone, we generated an AAVlibrary based on the liver-detargeted AAV9-N272A (PCT/US2017/068050) capgene platform that contained random 8-mer peptides with glycine-serinelinkers (5′-GGGS; 3′-GGGGS) substituted at the position Q588 in the AAV9capsid. In vivo selection in a specific cell type (e.g. neurons) wasperformed by recovering capsid sequences as antisense cap ORF mRNA frombrain tissue by RT-PCR. This method ensures that recovered sequences areonly derived from AAV variants that are capable of mediating RNAexpression in infected cells of our interest. When the hSynlenhancer-promoter is used, only sequences of AAV capsids that arecapable of transducing neurons can be retrieved, thus enablingneuron-specific selection of AAV capsids.

We first tested the ability of the TRADE system to recover the sequenceof the AAV cap gene from cell type-specific antisense mRNA using anAAV-PHP.B-hSynl-GFP-TRADE vector (FIG. 3 ). A hSynlenhancer-promoter-driven GFP expression cassette was incorporated in theAAV-PHP.B capsid gene-containing AAV vector genome in the TRADEconfiguration (FIG. 3A). This vector genome was packaged into theAAV-PHP.B capsid, and the resulting AAV vector was injectedintravenously into two 8-week-old male C57BL/6J mice (FIG. 3B). Twelvedays after injection, brain tissue was harvested. Tissue fixed with 4%paraformaldehyde was analyzed by immunofluorescence microscopy. Unfixedtissue was utilized for RNA extraction and RT-PCR analysis. We confirmedthat eGFP was expressed only in neurons (FIGS. 3C and 3D), indicatingthat the antisense mRNA transcribed from the cap gene is expressed in acell type-specific manner. We recovered antisense mRNA of the cap geneefficiently by RT-PCR (FIG. 3E). Sanger sequencing of a splice junctionunique to the antisense mRNA confirmed that RT-PCR products were indeedderived from the hSynl enhancer-promoter-driven antisense mRNA (FIG.3F). In addition, Sanger sequencing confirmed the sequence of the PHP.Bpeptide insertion (FIG. 3F). Together, these observations establishedthe ability of the TRADE system to successfully recover the AAV capsequence from the hSynl enhancer-promoter-driven antisense mRNAexpressed in AAV vector-transduced brain neurons.

With the successful establishment of the TRADE system, we performed twoAAV capsid directed evolution experiments; one used 8-week-old maleC57BL/6J mice and the other used one 8-month-old male rhesus macaque. Weproduced an AAV9-N272A-hSynl-GFP-TRADE-Lib library composed ofAAV9-derived mutant capsids that have a GGGS(N₈)GGGGS (SEQ ID NO:2)peptide insertion at the position of Q588 where N₈ represents a random8-mer peptide encoded by (NNK)₈. For the peptide insertion, Q588 wassubstituted with each peptide sequence. The diversity of the AAV librarywas at least 10⁷. In the mouse directed evolution experiment, we infusedthe AAV library via the tail vein at a dose of 3 x 10¹¹ vector genomes(vg) per mouse. For the second round of selection, we injected the AAVlibrary at a dose of 1×10¹², 1×10¹¹, 1×10¹⁰, or 1x10⁹ vector genomes(vg) using two mice. For the third round of selection, we injected theAAV library at a dose of 1×10¹¹ vg using two mice. We harvested braintissues twelve days after injection, and separated them into threeregions, i.e., the cerebrum, the cerebellum and the brain stem. Only thecerebrum samples were used for the directed evolution experiments. Weextracted total RNA from the cerebrum, reverse-transcribed the RNA usingan oligo dT primer, and amplified the peptide region including theflanking regions by a pair of the cap gene-specific PCR primers. TheRT-PCR products were then used to create the nextAAV9-N272A-hSynl-TRADE-Lib plasmid library, which was subsequently usedto produce the next AAV9-N272A-hSynl-TRADE-Lib virus library. For thesecond and third round selection, we packaged anAAV9-N272A-hSynl-TRADE-Lib genome that was devoid of the GFP ORF. In thenon-human primate directed evolution experiment, we infused theAAV9-N272A-hSynl-GFP-TRADE-Lib library via the saphenous vein at a doseof 2.0 x 10¹² vg per kg. Twelve days post-injection, the whole brain washarvested and sliced using a brain matrix, treated with RNAlater (ThermoFisher Scientific), and stored frozen. Total RNA was then extracted fromthe following brain regions: frontal cortex, occipital cortex,cerebellum (Purkinje and granular layers), medulla, pons, frontalcortex, hypothalamus, thalamus, cingulate gyrus, caudate nucleus,putamen, hippocampus, and preoptic area. We retrieved the peptidesequences by RT-PCR in the same manner as described above except that weperformed nested PCR to obtain PCR products sufficient for thedownstream Illumina and Sanger sequencing procedures. For some samples,we cloned the first PCR products directly into a plasmid backbonewithout performing nested PCR for Sanger sequencing. Following threerounds of selection in mice (Table 1) and one round of selection innon-human primate, we identified a number of potentiallytransduction-enhancing peptides inserted into the AAV9 capsids (Table2). We then generated a barcoded AAV library and utilized DNA/RNABarcode-Seq technology, previously developed in the Nakai lab (Adachi etal. Nat Commun 5, 3075 (2014); and PCT/US2017/068050), to compare thetransduction efficiency, tropism/biodistribution, and pharmacokineticsof 26 selected novel AAV variants (Table 3) following intravenousadministration in two commonly used mouse lines (C57BL/6J and BALB/cJ)and one rhesus macaque. As a result, we have found: (1) Some of thenovel variants identified by TRADE technology, in particularAAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2), performed up to8 times better than AAV9 in the brain of C57BL/6J mice (FIGS. 14B and14C). For HNx designation, please refer to Table 3. (2) As previouslyreported by Hordeaux et al. (Hordeaux et al. 2018), AAV-PHP.B transducedthe brain of BALB/cJ mice only at a level comparable to or lower thanthat of AAV9 (FIGS. 14B and 14C), demonstrating a mouse straindependency for AAV-PHP.B’s robust neurotropic enhancement. (3) Incontrast, AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2)retained robust neuronal transduction in BALB/cJ mice showing up to 7times better transduction than AAV9 (FIG. 14B). (4) In a rhesus macaque,many of the novel AAV mutants showed enhanced neuronal transduction, upto 4-fold greater than AAV9 in certain brain regions, while AAV-PHP.Btransduced non-human primate brain similarly to or lower than AAV9. Inparticular, AAV9-N272A-TTNLAKNS (HN1) transduced the non-human primatebrain best in multiple brain regions (FIGS. 14C and 14D). (5) All of theAAV9-N272A-derived variants including HN1, HN2 and HN3 showed varyingdegrees of liver-detargeting properties in mice and rhesus macaques(FIG. 17A). (6) AAV9-N272A-TTNLAKNS (HN1) and AAV9-N272A-QQNGTRPS (HN2)can transduce cells with the hSynl enhancer-promoter transcriptionalactivity in the lung up to 17 times better than AAV9 in mice (FIG. 17B,Tables 4 and 6). (7) AAV9-N272A-TTNLAKNS (HN1) exhibits vector genomedissemination to peripheral organs to a lesser degree compared to AAV9(FIGS. 17C and 17D). The AAV Barcode-Seq data are summarized in Tables 4to 9. Representative data presented in Tables 4 to 9 are also shown in agraph format in FIG. 14 and FIG. 17 .

TABLE 1 Peptide sequences identified by the hSynl-TRADE system using anAAV9-N272N-GGGS(Ns)GGGGS library targeting mouse brain neurons. 1stround 2nd round 3rd round ADKPPGLS SEQ ID NO:3 APTNFAHP SEQ ID NO:97AGAAYTPA (2) SEQ ID NO:150 AGEDGSSR SEQ ID NO:4 AQTNLAAG SEQ ID NO:98APSVSREK (2) SEQ ID NO:151 ALGTATQR SEQ ID NO:5 ASLPNLGQ SEQ ID NO:99DYMHKTGL SEQ ID NO:152 ALNTALVE SEQ ID NO:6 DYMHNTGL SEQ ID NO:100EEDAQLLI (2) SEQ ID NO:14 AMVRLTHN SEQ ID NO:7 DYMHTTGL SEQ ID NO:101ENKSAPLP SEQ ID NO:18 ASRDPSAT SEQ ID NO:8 ERNAWHAG SEQ ID NO:102GDYTVQRP SEQ ID NO:107 DANDARQR SEQ ID NO:9 ETQATPMP SEQ ID NO:103GGMNETTR SEQ ID NO:153 DLARMAAA SEQ ID NO:10 EWEDSARS SEQ ID NO:104GGSAFVTG SEQ ID NO:154 DQGSITAH SEQ ID NO:11 FTGDTDTL SEQ ID NO:105GGSPLAHP SEQ ID NO:21 DRTPGVNV SEQ ID NO:12 FTNRTSTT SEQ ID NO:106GNSHTGSS SEQ ID NO:155 DTDTLSPG SEQ ID NO:13 GDYTVQRP SEQ ID NO:107GPQEGSER (2) SEQ ID NO:109 EEDAQLLI SEQ ID NO:14 GGLRTDYG SEQ ID NO:108GQRGLPIA SEQ ID NO:27 EKLNDWPT SEQ ID NO:15 GGSPLAHP SEQ ID NO:21GSNHTQSL SEQ ID NO:110 ELNSARQV SEQ ID NO:16 GKQPVQPY SEQ ID NO:24HQVTSSGA (4) SEQ ID NO:33 ELQSFAGL SEQ ID NO:17 GPQEGSER SEQ ID NO:109LEQQRGAS SEQ ID NO:113 ENKSAPLP SEQ ID NO:18 GSNHTQSL SEQ ID NO:110LERNRDSD SEQ ID NO:39 ERTAVKGN SEQ ID NO:19 GTPQTTKE SEQ ID NO:29LLVTARSH (3) SEQ ID NO:44 GGIQTWT SEQ ID NO:20 HDRDTRQA SEQ ID NO:111MESQRANS (2) SEQ ID NO:117 GGSPLAHP SEQ ID NO:21 LDQNRRPQ SEQ ID NO:112MSGQGYQA (2) SEQ ID NO:50 GGTAAQGV SEQ ID NO:22 LEQQRGAS SEQ ID NO:113NSARTQLS SEQ ID NO:156 GKMASGSL SEQ ID NO:23 LERNRDSD SEQ ID NO:39PLTILNRH SEQ ID NO:157 GKQPVQPY SEQ ID NO:24 LGGNAQGL SEQ ID NO:114QGTRTNPP SEQ ID NO:158 GNPHTGST SEQ ID NO:25 LLVTTRSH SEQ ID NO:115QQNGTRPS (4) SEQ ID NO:128 GPTLGGSG SEQ ID NO:26 LVTNTTR SEQ ID NO:116QSGDSALN (3) SEQ ID NO:67 GQRGLPIA SEQ ID NO:27 MESQRANS SEQ ID NO:117QSSAMPRN (2) SEQ ID NO:159 GREPRRLH SEQ ID NO:28 MISQTLMA SEQ ID NO:118SATISLQV SEQ ID NO:136 GTPQTTKE SEQ ID NO:29 MMSQSLRA SEQ ID NO:119SHNSQPVA SEQ ID NO:160 GVTERPNR SEQ ID NO:30 NNVQSALN SEQ ID NO:120SHTNLRDT SEQ ID NO:137 HLGDNLAR SEQ ID NO:31 NSARTQLS SEQ ID NO:121SSGYLTAN SEQ ID NO:139 HPGSGAGP SEQ ID NO:32 PQWNRTPL SEQ ID NO:122TAQGAAFR (4) SEQ ID NO:161 HQVTSSGA SEQ ID NO:33 PRFNNSSL SEQ ID NO:123TPGLNNAR SEQ ID NO:162 HVGSQMHA SEQ ID NO:34 PRPTWGT SEQ ID NO:60TSLGTPEA SEQ ID NO:163 IG*TVPMQ SEQ ID NO:35 PVDGGRHL SEQ ID NO:124TTNLAKNS (6) SEQ ID NO:164 KFTRDGPY SEQ ID NO:36 PWFNKSSL SEQ ID NO:125WQGEQKR (4) SEQ ID NO:146 KGPAEQGH SEQ ID NO:37 QDMNSQRS SEQ ID NO:126WSPDAVEG SEQ ID NO:165 LAHSPRLW SEQ ID NO:38 QGASNSQL SEQ ID NO:127WSQDAVKG (2) SEQ ID NO:148 LERNRDSD SEQ ID NO:39 QQNGTRPS SEQ ID NO:128WTGGGSGT (3) SEQ ID NO:149 LETHTSLT SEQ ID NO:40 QRSAYPTS SEQ ID NO:129WTGGRHL SEQ ID NO:166 LHDGKYST SEQ ID NO:41 QRTPSITP SEQ ID NO:130LKATGRGK SEQ ID NO:42 QWMKEQAG SEQ ID NO:131 LLPGSADG SEQ ID NO:43RDGRHPSE SEQ ID NO:132 LLVTARSH SEQ ID NO:44 RGTVTVEQ SEQ ID NO:133LPEVEPTN SEQ ID NO:45 RPANHSTA SEQ ID NO:134 LPWENSSQ SEQ ID NO:46RQGDADTL SEQ ID NO:135 LQRNSDAN SEQ ID NO:47 SATISLQV SEQ ID NO:136LQSAPRAT SEQ ID NO:48 SHTNLRDT SEQ ID NO:137 MLGSQVPT SEQ ID NO:49SRMGETPQ SEQ ID NO:138 MSGQGYQA SEQ ID NO:50 SSGYLTAN SEQ ID NO:139NPGRDFRD SEQ ID NO:51 SSWSQGP SEQ ID NO:79 NQPSDYVS SEQ ID NO:52TGNSPEQA SEQ ID NO:140 NSVGSADK SEQ ID NO:53 THSQGRLA SEQ ID NO:141NVQRTQRG SEQ ID NO:54 TPIVGSNV SEQ ID NO:142 PAQLNGPR SEQ ID NO:55TPPKSPSM SEQ ID NO:143 PERERLPR SEQ ID NO:56 TRMDERSP SEQ ID NO:144PGNGSHTM SEQ ID NO:57 TTATTSIT SEQ ID NO:145 PIPGTPQP SEQ ID NO:58WQGEQKR SEQ ID NO:146 PMSVPASN SEQ ID NO:59 WNDRSGER SEQ ID NO:147PRPTWGT SEQ ID NO:60 WSQDAVKG SEQ ID NO:148 PRTNRGPE SEQ ID NO:61WTGGGSGT SEQ ID NO:149 PVANPTTA SEQ ID NO:62 PVLGGPPK SEQ ID NO:63QGSRQGSS SEQ ID NO:64 QMAETPIS SEQ ID NO:65 QMLGIGRS SEQ ID NO:66QSGDSALN SEQ ID NO:67 RAGLTSSE SEQ ID NO:68 RLDNTGVG SEQ ID NO:69RMPGKPYS SEQ ID NO:70 RVAGASQP SEQ ID NO:71 RVESSQLE SEQ ID NO:72SARTGASE SEQ ID NO:73 SERNRASM SEQ ID NO:74 SIDVRMAA SEQ ID NO:75SRDGHILR SEQ ID NO:76 SRQWLPG SEQ ID NO:77 SSRGYTST SEQ ID NO:78 SSWSQGPSEQ ID NO:79 SVAESGRE SEQ ID NO:80 TALTANTQ SEQ ID NO:81 TESSVGNL SEQ IDNO:82 TGREGANL SEQ ID NO:83 TLSEPPKK SEQ ID NO:84 TNAVSGKS SEQ ID NO:85TRAPTIHL SEQ ID NO:86 TRESTDRG SEQ ID NO:87 TVAAAPNL SEQ ID NO:88TYHNNTPR SEQ ID NO:89 VSNSTRTS SEQ ID NO:90 VTLQIDTK SEQ ID NO:91WMSRPGPT SEQ ID NO:92 WPYRGLTQ SEQ ID NO:93 WRRQGSRA SEQ ID NO:94YAQRFAKM SEQ ID NO:95 YNSPRQTV SEQ ID NO:96 The table lists peptideinsertions on AAV9-N272A after each of three rounds of selection. Thenumbers in parentheses indicate the frequency of each peptide among atotal of 69 peptides identified after the three round of selection.Peptides with no number were found only once. The sequences of thepeptide region were determined by Sanger sequencing. Actual peptidesequences were randomized octapeptides flanked by glycine-serine linkerssuch that position Q588 was substituted with GGGS(N₈)GGGGS. For example,“-TNHQSAGGGSTTNLAKNSGGGGSAQAQTG-” for TTNLAKNS and“-TNHQSAGGGSQQNGTRPSGGGGSAQAQTG-” for QQNGTRPS.

TABLE 2 Peptide sequences identified by the hSynl-TRADE system using anAAV9-N272A-GGGS(N_(s))GGGGS library targeting rhesus macaque brainneurons 1st round AVAGDRLL SEQ ID NO:167 DLLTRSVS SEQ ID NO:168 EWKTQLALSEQ ID NO:169 GNINWPH SEQ ID NO:170 GSPAASSW SEQ ID NO:171 KHSLTLES SEQID NO:172 KPVSTDTF SEQ ID NO:173 LDRSGSTG SEQ ID NO:174 LGAQNHVV SEQ IDNO:175 LMATDYGP SEQ ID NO:176 LRATDYGP SEQ ID NO:177 MERTEPLG SEQ IDNO:178 NDGLRLHL SEQ ID NO:179 NLSAHSHA SEQ ID NO:180 NLSAHSHD SEQ IDNO:181 RALDLVTR SEQ ID NO:182 SAGMARNS SEQ ID NO:183 SGQRVGSA SEQ IDNO:184 SGQRVGSD SEQ ID NO:185 TAQGAAFR SEQ ID NO:161 TGRPEQPK SEQ IDNO:186 THSPIKLP SEQ ID NO:187 TQFSQAQR SEQ ID NO:188 VGDSANLR SEQ IDNO:189 The sequences of the peptide region were determined either byIllumina sequencing or Sanger sequencing. Actual peptide sequences wererandomized octapeptides flanked by glycine-serine linkers such thatposition Q588 was substituted with GGGS(N₈)GGGGS. These peptides wererecovered from frontal cortex, occipital cortex, hypothalamus andthalamus.

TABLE 3 A list of the 29 AAV capsids contained in the DNA/RNA-barcodeddsAAV-hSynl-GFP-BCLib library used for phenotype determination of eachAAV strain AAV strain (AAV capsid) Abbreviation Number of viral clonesin the AAV library Note AAV9 AAV9 15 Reference AAV9-N272A AAV9-N272A 5Reference AAV-PHP.B AAV-PHP.B 2 Reference AAV9-N272A-TTNLAKNS (peptideinsertion site SEQ ID NO:164) HN1 2 TRADE variant (C57BL/6J)AAV9-N272A-QQNGTRPS (peptide insertion site SEQ ID NO:128) HN2 2 TRADEvariant (C57BL/6J) AAV9-N272A-SGQRVGSD (peptide insertion site SEQ IDNO:185) HN3 2 TRADE variant (rhesus macaque) AAV9-N272A-AVAGDRLL(peptide insertion site SEQ ID NO:167) HN4 2 TRADE variant (rhesusmacaque) AAV9-N272A-DLLTRSVS (peptide insertion site SEQ ID NO:168) HN52 TRADE variant (rhesus macaque) AAV9-N272A-EWKTQLAL (peptide insertionsite SEQ ID NO:169) HN6 2 TRADE variant (rhesus macaque)AAV9-N272A-GNINVVPH (peptide insertion site SEQ ID NO:170) HN7 2 TRADEvariant (rhesus macaque) AAV9-N272A-GSPAASSW (peptide insertion site SEQID NO:171) HN8 2 TRADE variant (rhesus macaque) AAV9-N272A-KHSLTLES(peptide insertion site SEQ ID NO:172) HN9 2 TRADE variant (rhesusmacaque) AAV9-N272A-KPVSTDTF (peptide insertion site SEQ ID NO:173) HN102 TRADE variant (rhesus macaque) AAV9-N272A-LDRSGSTG (peptide insertionsite SEQ ID NO:174) HN11 2 TRADE variant (rhesus macaque)AAV9-N272A-LGAQNHVV (peptide insertion site SEQ ID NO:175) HN12 2 TRADEvariant (rhesus macaque) AAV9-N272A-LRATDYGP (peptide insertion site SEQID NO:177) HN13 2 TRADE variant (rhesus macaque) AAV9-N272A-MERTEPLG(peptide insertion site SEQ ID NO:178) HN14 2 TRADE variant (rhesusmacaque) AAV9-N272A-NDGLRLHL (peptide insertion site SEQ ID NO:179) HN152 TRADE variant (rhesus macaque) AAV9-N272A-NLSAHSHD (peptide insertionsite SEQ ID NO:181) HN16 2 TRADE variant (rhesus macaque)AAV9-N272A-RALDLVTR (peptide insertion site SEQ ID NO:182) HN17 2 TRADEvariant (rhesus macaque) AAV9-N272A-SAGMARNS (peptide insertion site SEQID NO:183) HN18 2 TRADE variant (rhesus macaque) AAV9-N272A-TAQGAAFR(peptide insertion site SEQ ID NO:161) HN19 2 TRADE variant (rhesusmacaque) AAV9-N272A-TGRPEQPK (peptide insertion site SEQ ID NO:186) HN202 TRADE variant (rhesus macaque) AAV9-N272A-THSPIKLP (peptide insertionsite SEQ ID NO:187) HN21 2 TRADE variant (rhesus macaque)AAV9-N272A-TQFSQAQR (peptide insertion site SEQ ID NO:188) HN22 2 TRADEvariant (rhesus macaque) AAV9-N272A-VGDSANLR (peptide insertion site SEQID NO:189) HN23 2 TRADE variant (rhesus macaque) AAV9-N272A-HQVTSSGA(peptide insertion site SEQ ID NO:33) HN24 2 TRADE variant (mouse)AAV9-N272A-LLVTARSH (peptide insertion site SEQ ID NO:44) HN25 2 TRADEvariant (mouse) AAV9-N272A-VVQGEQKR (peptide insertion site SEQ IDNO:146) HN26 2 TRADE variant (mouse) The novel AAV9-hSynl-TRADE-derivedcapsid variants were selected from those identified following threerounds of selection in mice (Table 1) and one round of selection in arhesus macaque (Table 2). Each recovered AAV variant was assigned anabbreviation, HNx. A DNA/RNA-barcoded dsAAV-hSynl-GFP-BCLib librarycontaining was constructed such that each AAV variant packaged a uniquedsAAV-hSynl-GFP-BC viral genome expressing AAV variant-specific RNAbarcodes. The number of unique AAV barcode clones for each variant,including critical reference variants, is presented in this table.

TABLE 4 Brain neuronal transduction efficiency and biodistribution ofthe TRADE-identified AAV variants in C57BL/6J mice following intravenousadministration Brain (RNA) Lung (RNA) Heart (DNA) Kidney (DNA) Liver(DNA) Lung (DNA) Muscle (DNA) Pancreas (DNA) Spleen (DNA) Testis (DNA)AAV9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 AAV9-N272A 1.930.22 0.82 2.11 0.06 0.51 0.17 0.25 4.49 1.42 AAV-PHP.B 9.42 0.64 0.922.35 0.27 0.81 0.36 0.62 2.69 1.45 HN1 8.01 2.74 0.21 0.62 0.24 3.400.36 0.42 0.41 0.64 HN2 6.52 1.19 0.36 0.56 0.24 2.55 0.32 0.33 0.660.91 HN3 2.35 0.41 0.95 1.29 0.09 0.45 0.20 0.25 2.57 1.45 HN4 0.87 0.150.65 2.96 0.02 0.48 0.11 0.21 4.02 1.22 HN5 0.46 0.10 0.57 2.66 0.010.47 0.09 0.21 3.90 1.10 HN6 0.37 0.22 0.64 3.13 0.01 0.52 0.08 0.274.27 1.22 HN7 1.03 0.21 0.40 0.54 0.25 0.17 0.23 0.10 0.75 0.43 HN8 0.740.11 0.61 2.61 0.02 0.45 0.09 0.16 3.27 1.06 HN9 1.47 0.28 0.62 1.490.08 0.30 0.15 0.16 2.41 0.82 HN10 1.40 0.18 0.64 1.48 0.04 0.24 0.120.16 2.64 0.97 HN11 1.38 0.21 0.73 1.37 0.05 0.29 0.15 0.17 3.17 1.12HN12 0.80 0.17 0.26 0.42 0.24 0.16 0.20 0.06 0.49 0.21 HN13 1.59 0.280.77 1.17 0.10 0.28 0.19 0.18 2.28 0.93 HN14 0.45 0.05 0.47 1.31 0.010.20 0.07 0.14 1.86 0.65 HN15 0.50 0.21 0.68 3.48 0.01 0.58 0.10 0.244.70 1.30 HN16 1.43 0.24 0.58 1.28 0.02 0.32 0.11 0.22 3.70 1.07 HN170.29 0.07 0.50 2.80 0.01 0.46 0.08 0.19 3.73 1.05 HN18 1.46 0.12 0.681.78 0.11 0.28 0.17 0.14 2.10 0.92 HN19 0.56 0.10 0.57 3.07 0.01 0.520.09 0.18 3.98 1.16 HN20 1.68 0.35 0.90 1.10 0.18 0.29 0.24 0.18 1.890.89 HN21 0.26 0.08 0.50 2.53 0.01 0.46 0.06 0.14 3.44 1.01 HN22 0.820.06 0.51 2.55 0.02 0.42 0.10 0.19 3.32 1.05 HN23 2.45 0.26 0.72 1.020.05 0.30 0.15 0.21 2.25 1.12 HN24 1.33 0.22 0.63 1.31 0.05 0.28 0.140.15 2.83 0.90 HN25 0.37 0.12 0.64 3.25 0.02 0.53 0.09 0.22 4.35 1.23HN26 0.73 0.14 0.63 2.53 0.06 0.40 0.12 0.17 2.86 1.07 ADNA/RNA-barcoded dsAAV-hSynl-GFP-BC library (dsAAV-hSynl-GFP-BCLib)containing 26 novel AAV variants identified by TRADE and control AAVcapsids was injected intravenously into 3 C57BL/6J mice at a dose of 5 x10¹¹ vg per mouse (for the library, see Table 3). Two weekspost-injection, various tissues were harvested and analyzed for braintransduction by AAV RNA Barcode-Seq and biodistribution to peripheralorgans by AAV DNA Barcode-Seq. All the values are normalized with thoseof AAV9 (AAV9=1.0).

TABLE 5 Pharmacokinetic profiles of TRADE-identified AAV variants inC57BL/6J mice following intravenous administration 1 m 10 m 30 m 1 h 4 h8 h 24 h 72 h AAV9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 AAV9-N272A1.16 1.17 1.28 1.34 1.77 2.02 3.14 0.13 AAV-PHP.B 1.38 1.42 1.44 1.621.88 2.15 3.14 0.39 HN1 0.87 0.47 0.29 0.29 0.27 0.31 0.37 0.02 HN2 0.790.60 0.49 0.55 0.59 0.66 0.91 0.03 HN3 1.12 1.15 1.21 1.32 1.65 1.883.05 0.04 HN4 1.32 1.50 1.43 1.52 2.01 2.37 3.78 0.03 HN5 1.14 1.30 1.371.42 1.84 2.03 3.37 0.02 HN6 1.21 1.34 1.43 1.60 1.99 2.38 3.90 0.02 HN70.92 0.88 0.93 0.93 1.09 1.21 1.22 0.03 HN8 1.20 1.29 1.30 1.44 1.832.10 3.42 0.03 HN9 0.94 0.91 0.97 0.99 1.26 1.36 1.97 0.04 HN10 0.980.98 1.00 1.06 1.31 1.36 1.89 0.02 HN11 1.00 1.04 1.04 1.14 1.39 1.482.11 0.02 HN12 0.93 0.93 0.84 0.79 0.71 0.61 0.62 0.01 HN13 0.95 0.900.95 0.95 1.20 1.28 1.60 0.03 HN14 0.94 0.95 1.00 1.08 1.41 1.58 2.560.01 HN15 1.39 1.56 1.67 1.66 2.20 2.76 4.26 0.03 HN16 0.98 1.00 1.041.15 1.46 1.63 2.77 0.02 HN17 1.32 1.28 1.27 1.31 1.94 2.13 4.03 0.04HN18 1.10 1.06 0.96 0.93 0.82 0.84 1.27 0.01 HN19 1.39 1.39 1.51 1.492.04 2.50 4.09 0.03 HN20 1.15 1.09 1.19 1.14 1.41 1.70 1.97 0.06 HN211.25 1.19 1.34 1.38 1.90 1.99 3.30 0.02 HN22 1.16 1.24 1.32 1.35 1.742.13 3.74 0.02 HN23 1.03 1.02 1.04 1.14 1.43 1.64 2.56 0.03 HN24 0.991.01 1.05 1.16 1.45 1.58 2.38 0.03 HN25 1.29 1.40 1.44 1.49 1.93 2.493.74 0.03 HN26 1.21 1.19 1.29 1.30 1.74 2.03 3.09 0.03 AAV DNABarcode-Seq analysis was performed on the blood samples obtained fromthe mice injected with 1 x 10¹³ vg/kg of the DNA/RNA-barcodeddsAAV-hSynl-GFP-BCLib library (see Table 3, n=2) All the values arenormalized with those of AAV9 (AAV9=1.0). All the values are normalizedto AAV9 (AAV9=1.0).

TABLE 6 Brain neuronal transduction efficiency and biodistribution ofthe TRADE-identified AAV variants in BALB/cJ mice following intravenousadministration. Brain (RNA) Lung (RNA) Heart (DNA) Kidney (DNA) Liver(DNA) Lung (DNA) Muscle (DNA) Pancreas (DNA) Spleen (DNA) Testis (DNA)AAV9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 AAV9-N272A 1.000.30 0.45 1.23 0.03 0.64 0.29 1.23 2.75 0.17 AAV-PHP.B 1.06 0.44 0.561.30 0.22 0.67 0.40 1.12 1.69 0.30 HN1 7.59 17.84 0.18 0.52 0.05 3.750.21 0.28 0.31 0.36 HN2 3.26 4.71 0.31 0.52 0.07 3.13 0.25 0.39 0.690.44 HN3 1.00 0.35 0.51 1.04 0.04 0.41 0.20 0.60 1.38 0.14 HN4 0.41 0.410.29 1.58 0.01 0.44 0.16 1.18 2.50 0.09 HN5 0.28 0.15 0.25 1.34 0.000.38 0.13 1.05 2.25 0.07 HN6 0.25 0.06 0.30 1.60 0.01 0.45 0.17 1.172.69 0.07 HN7 0.54 0.21 0.27 0.65 0.12 0.22 0.16 0.32 0.75 0.11 HN8 0.340.03 0.28 1.53 0.01 0.38 0.14 0.94 2.10 0.07 HN9 0.49 0.19 0.28 1.180.02 0.22 0.11 0.45 1.08 0.07 HN10 0.54 0.19 0.33 1.25 0.01 0.30 0.140.50 1.82 0.08 HN11 0.23 0.12 0.23 1.15 0.03 0.17 0.09 0.62 0.95 0.05HN12 0.34 0.13 0.19 0.98 0.03 0.12 0.07 0.23 0.48 0.04 HN13 0.43 0.250.36 1.25 0.03 0.25 0.15 0.41 1.01 0.09 HN14 0.22 0.08 0.20 0.93 0.020.14 0.08 0.38 0.68 0.04 HN15 0.25 0.26 0.33 1.80 0.01 0.46 0.19 1.393.03 0.10 HN16 0.62 0.25 0.32 0.93 0.01 0.41 0.16 0.71 1.55 0.10 HN170.18 0.12 0.22 1.40 0.00 0.37 0.13 0.96 2.29 0.07 HN18 0.75 0.16 0.401.46 0.04 0.29 0.15 0.59 1.36 0.08 HN19 0.28 0.10 0.28 1.57 0.01 0.440.15 1.14 2.51 0.08 HN20 0.69 0.11 0.42 0.56 0.08 0.31 0.13 0.53 0.850.12 HN21 0.14 0.15 0.25 1.38 0.00 0.34 0.11 0.91 2.34 0.08 HN22 0.410.09 0.22 1.33 0.01 0.36 0.12 1.01 2.09 0.07 HN23 0.79 0.32 0.33 0.990.02 0.36 0.17 0.43 1.24 0.10 HN24 0.56 0.25 0.34 1.17 0.02 0.34 0.140.53 1.33 0.08 HN25 0.19 0.02 0.30 1.65 0.01 0.49 0.17 1.15 2.68 0.09HN26 0.31 0.11 0.33 1.52 0.02 0.35 0.17 0.84 1.77 0.09 ADNA/RNA-barcoded dsAAV-hSynl-GFP-BC library (dsAAV-hSynl-GFP-BCLib)containing 26 novel AAV variants identified by TRADE and control AAVcapsids was injected intravenously into 3 BALB/cJ mice at a dose of 5 x10¹¹ vg per mouse (for the library, see Table 3). Two weekspost-injection, various tissues were harvested and analyzed for braintransduction by AAV RNA Barcode-Seq and biodistribution to peripheralorgans by AAV DNA Barcode-Seq. All the values are normalized with thoseof AAV9 (AAV9=1.0).

TABLE 7 Transduction efficiency of hSynl-TRADE-derived AAV variants invarious brain regions of one rhesus macaque following intravenousadministration as determined by AAV hSynl-RNA Barcode-Seq analysisCerebellu (Granular layer) Cerebellum (Purkinje CingulateGyrus FrontalCortex Hippocampus Hypothalamus Medulla Occipital Cortex Pons PreopticArea Putamen Thalamus AAV9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.001.00 1.00 1.00 AAV9-N272A 1.28 1.63 1.76 1.75 2.08 1.00 1.45 1.51 1.450.66 1.99 1.19 AAV-PHP.B 0.78 1.21 1.27 1.50 1.39 0.94 1.33 1.23 1.620.35 1.29 0.69 HN1 1.99 1.78 2.68 2.18 4.07 2.05 1.39 1.54 1.99 1.992.54 2.84 HN2 0.81 0.96 1.06 1.13 1.45 0.78 0.77 0.84 0.79 0.72 1.151.16 HN3 1.75 2.03 2.14 2.28 3.54 1.48 1.87 1.67 1.71 1.28 2.20 1.87 HN40.27 0.61 0.70 0.68 0.37 0.39 0.58 0.54 0.27 0.30 0.49 0.62 HN5 0.150.27 0.35 0.22 0.15 0.01 0.27 0.17 0.27 0.22 0.31 0.15 HN6 0.06 0.280.36 0.12 0.10 0.27 0.04 0.12 0.12 0.01 0.26 0.10 HN7 0.96 1.40 1.441.45 1.64 1.13 1.48 1.18 1.01 0.87 1.39 1.29 HN8 0.38 0.61 0.59 0.670.73 0.42 0.56 0.42 0.45 0.35 0.57 0.50 HN9 1.17 1.45 1.91 1.66 2.150.92 1.65 1.30 1.27 1.10 1.96 1.48 HN10 1.08 1.24 1.40 1.45 1.78 0.871.33 1.16 1.11 0.76 1.50 1.00 HN11 0.96 1.22 1.37 1.42 1.62 1.00 1.281.15 1.12 0.65 1.41 1.19 HN12 1.04 1.43 1.64 1.70 1.98 0.97 1.49 1.261.09 0.73 1.74 1.44 HN13 1.77 1.74 1.86 1.82 2.17 1.20 2.38 1.54 1.971.36 2.14 1.77 HN14 0.13 0.45 0.27 0.26 0.28 0.14 0.30 0.32 0.19 0.040.23 0.17 HN15 0.38 0.43 0.19 0.46 0.44 0.04 0.28 0.19 0.23 0.63 0.360.09 HN16 0.57 0.65 0.79 0.82 0.89 0.35 0.75 0.77 0.64 0.18 0.72 0.54HN17 0.05 0.18 0.24 0.14 0.08 0.28 0.16 0.11 0.07 0.01 0.19 0.03 HN181.21 1.23 1.62 1.70 2.60 1.17 1.47 1.15 1.09 0.69 1.46 1.13 HN19 0.240.24 0.21 0.59 0.50 0.13 0.14 0.26 0.26 0.01 0.31 0.17 HN20 1.08 1.421.60 1.81 2.28 1.45 1.51 1.22 1.41 1.32 2.27 1.34 HN21 0.19 0.11 0.050.15 0.04 0.49 0.27 0.19 0.08 0.01 0.10 0.14 HN22 0.27 0.17 0.48 0.590.49 0.24 0.24 0.27 0.19 0.01 0.23 0.12 HN23 0.60 1.01 1.21 1.11 1.550.61 1.23 0.92 0.95 0.49 1.25 0.76 HN24 0.99 1.18 1.19 1.33 1.71 0.701.17 1.06 1.04 0.57 1.39 1.21 HN25 0.13 0.14 0.06 0.32 0.21 0.12 0.230.24 0.07 0.01 0.28 0.08 HN26 0.35 0.52 0.42 0.60 0.88 0.40 0.50 0.440.36 0.27 0.61 0.28 AAV RNA Barcode-Seq analysis was performed on RNAsextracted from various brain regions of one rhesus macaque (n=1)intravenously injected with 2.0 x 10¹³ vg/kg of a DNA/RNA-barcodeddsAAV-hSynl-GFP-BCLib library that expresses RNA barcodes under thecontrol of the hSynl enhancer-promoter. All the values are normalizedwith those of AAV9 (AAV9=1.0).

TABLE 8 Pharmacokinetic profiles of hSynl-TRADE-derived AAV variants inrhesus macaque following intravenous administration. 1 m 10 m 30 m 1 h 4h 8 h 24 h 72 h AAV9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 AAV9-N272A0.95 0.96 1.06 1.05 1.24 1.79 2.64 1.18 AAV-PHP.B 1.00 1.10 1.16 1.111.27 1.99 2.94 1.69 HN1 0.78 0.66 0.71 0.65 0.67 0.88 0.65 0.30 HN2 0.690.72 0.69 0.62 0.75 0.92 1.24 0.25 HN3 0.85 0.86 0.96 0.77 1.12 1.702.60 0.60 HN4 1.07 1.07 1.15 1.15 1.30 1.88 3.39 2.15 HN5 0.99 0.98 1.141.06 1.19 1.62 2.83 1.39 HN6 1.13 1.09 1.18 1.09 1.32 1.97 3.32 1.90 HN70.75 0.78 0.81 0.79 0.95 1.33 1.78 0.27 HN8 1.05 1.06 1.02 1.07 1.271.82 3.02 1.67 HN9 0.83 0.76 0.87 0.78 0.92 1.36 1.91 0.29 HN10 0.840.87 0.90 0.87 1.14 1.32 2.32 0.28 HN11 0.88 0.89 0.90 0.86 1.20 1.552.42 0.61 HN12 0.81 0.80 0.86 0.80 0.99 1.41 1.90 0.27 HN13 0.76 0.710.82 0.76 0.90 1.31 1.86 0.29 HN14 0.76 0.75 0.83 0.78 0.99 1.40 2.210.18 HN15 1.31 1.07 1.27 1.23 1.36 2.37 4.46 2.03 HN16 0.76 0.80 0.910.84 0.98 1.41 1.90 0.26 HN17 1.02 1.07 1.21 1.00 1.29 1.88 2.91 1.84HN18 0.88 0.88 0.96 0.90 1.10 1.57 2.44 0.58 HN19 1.08 1.04 1.15 1.141.26 2.04 3.69 2.05 HN20 0.89 0.82 0.88 0.81 0.91 1.62 2.28 0.39 HN211.00 1.00 1.11 0.98 1.20 1.54 2.41 2.10 HN22 0.96 0.97 1.09 1.03 1.241.74 2.90 1.82 HN23 0.76 0.76 0.86 0.80 0.97 1.50 2.14 0.35 HN24 0.931.00 0.96 1.00 1.41 1.48 2.31 1.41 HN25 1.05 1.16 1.08 1.19 1.18 2.003.52 1.54 HN26 1.03 0.98 1.08 1.07 1.18 1.77 2.72 1.56 AAV DNABarcode-Seq analysis was performed on the blood samples obtained from asingle rhesus macaque injected with 2 x 10¹³ vg/kg of theDNA/RNA-barcoded dsAAV-hSynl-GFP-BCLib library (the same animal as inTable 7). All the values are normalized with those of AAV9 (AAV9=1.0).

TABLE 9 Biodistribution of hSynl-TRADE-derived AAV variants toperipheral tissues of a rhesus macaque following intravenousadministration as determined by AAV DNA Barcode-Seq analysis Liver HeartLung Kidney Pancreas Spleen Gastocnemius muscle oleus muscleS IntestineBone marrow Smooth muscle (stomach AAV9 1.00 1.00 1.00 1.00 1.00 1.001.00 1.00 1.00 1.00 1.00 AAV9-N272A 0.07 0.20 0.79 1.29 2.91 4.67 0.260.50 0.64 0.06 0.82 AAV-PHP.B 0.35 0.42 1.09 1.22 2.55 2.14 0.54 0.770.40 0.43 0.80 HN1 0.26 0.41 0.34 0.41 0.35 0.76 1.04 1.12 0.24 0.330.33 HN2 0.16 0.17 0.35 0.32 0.51 0.71 0.32 0.52 0.11 0.07 0.30 HN3 0.120.21 0.78 0.91 2.19 2.48 0.25 0.58 0.18 0.05 0.63 HN4 0.02 0.08 0.781.12 2.25 3.42 0.17 0.41 0.15 0.03 0.39 HN5 0.01 0.05 0.73 0.98 2.242.85 0.10 0.30 0.07 0.01 0.37 HN6 0.01 0.05 0.92 1.11 2.15 3.50 0.110.40 0.12 0.01 0.30 HN7 0.27 0.19 0.36 0.43 0.47 0.51 0.18 0.23 0.160.12 0.37 HN8 0.04 0.08 0.71 0.98 2.28 2.57 0.12 0.38 0.11 0.02 0.44 HN90.12 0.13 0.32 0.36 0.71 0.79 0.13 0.31 0.11 0.04 0.29 HN10 0.08 0.130.63 0.63 1.39 2.53 0.16 0.38 0.11 0.02 0.28 HN11 0.06 0.16 0.63 0.741.62 1.68 0.18 0.44 0.16 0.03 0.47 HN12 0.19 0.11 0.28 0.38 0.56 0.610.10 0.19 0.09 0.05 0.30 HN13 0.18 0.23 0.58 0.44 0.88 1.12 0.21 0.380.18 0.06 0.49 HN14 0.00 0.04 0.30 0.49 0.59 0.83 0.06 0.28 0.05 0.010.34 HN15 0.01 0.06 1.01 1.43 2.43 3.80 0.13 0.46 0.10 0.02 0.69 HN160.02 0.07 0.37 1.00 0.70 0.87 0.08 0.17 0.06 0.02 0.28 HN17 0.01 0.040.85 1.01 2.38 3.25 0.08 0.29 0.10 0.01 0.32 HN18 0.15 0.13 0.36 0.520.90 1.17 0.13 0.31 0.09 0.01 0.33 HN19 0.06 0.08 0.93 1.14 2.98 3.410.15 0.42 0.10 0.02 0.36 HN20 0.36 0.22 0.34 0.50 0.88 0.96 0.21 0.480.14 0.05 0.31 HN21 0.02 0.05 0.74 1.03 2.17 3.08 0.12 0.33 0.06 0.010.57 HN22 0.06 0.07 0.72 0.89 1.73 2.68 0.14 0.31 0.09 0.02 0.25 HN230.04 0.08 0.61 0.28 0.74 0.71 0.09 0.30 0.10 0.01 0.24 HN24 0.06 0.140.66 0.74 1.99 1.98 0.18 0.43 0.13 0.03 0.50 HN25 0.06 0.07 0.90 1.212.76 3.28 0.22 0.43 0.10 0.03 0.56 HN26 0.17 0.10 0.64 0.90 1.84 2.380.12 0.35 0.12 0.02 0.56 AAV DNA Barcode-Seq analysis was performed onDNA extracted from various peripheral tissues of one rhesus macaque(n=1, the same animal as presented in Table 7) intravenously injectedwith 2 x 10¹³ vg/kg of a DNA/RNA-barcoded dsAAV-hSynl-GFP-BCLib library.All values are normalized to AAV9 (AAV9=1.0).

TABLE 9 Splice donor and splice acceptor sites identified in antisenseAAV cap ORF transcripts. SEQ ID AAV serotype SD or SA Exon-intronjunction sequence (Introns are underlined) SEQ ID NO:199 AAV1 SD1009-CTTACCAGCA-1018 SEQ ID NO:199 AAV3 SD 1006-CTTACCAGCA-1015 SEQ IDNO:200 AAV1 SD 1228-TTTACCTTCA-1237 SEQ ID NO:201 AAV3 SD1237-TATACCTTCG-1246 SEQ ID NO:202 AAV1 SD 1331-ATTACCTGAA-1340 SEQ IDNO:203 AAV1 SD 1434-GCTACCTGGA-1443 SEQ ID NO:204 AAV1 SD1502-TTTACCTGGA-1510 SEQ ID NO:205 AAV1 SD 1803-ATTACCTGGC-1812 SEQ IDNO:206 AAV3 SD 1803-CTTACCTGGC-1812 SEQ ID NO:207 AAV1 SD1835-TGTACCTGCA-1844 SEQ ID NO:208 AAV1 SD 2189-GTTACCTTAC-2198 SEQ IDNO:209 AAV9 SD 2189-GATACCTGAC-2198 SEQ ID NO:210 AAV1 SD2194-CTTACCCGTC-2203 SEQ ID NO:211 AAV3 SD 2194-CTCACACGAA-2203 SEQ IDNO:212 AAV1 SA 305-AGCGTCTGCA-314 SEQ ID NO:213 AAV1 SA414-GGCTCCTGGA-423 SEQ ID NO:213 AAV3 SA 414-GGCTCCTGGA-423 SEQ IDNO:214 AAV1 SA 495-GCCCGCTAAA-504 SEQ ID NO:214 AAV9 SA495-GCCCGCTAAA-504 SEQ ID NO:215 AAV3 SA 1133-TCACCCTGAA-1142 SEQ IDNO:216 AAV1 SA 1181-ACTGCCTGGA-1190 SEQ ID NO:202 AAV1 SA1331-ATTACCTGAA-1340 SEQ ID NO:217 AAV3 SA 1328-ACTACCTGAA-1337 SEQ IDNO:218 AAV1 SA 1464-CGTTTCTAAA-1473 SEQ ID NO:219 AAV1 SA1653-AAACACTGCA-1662 SEQ ID NO:220 AAV1 SA 2054-GGGAGCTGCA-2063 SEQ IDNO:463 AAV3 SA 2054-GGGAGCTACA-2063 Ten nucleotides around exon-intronjunctions identified in antisense AAV cap mRNA are presented with thejunction at the center. Letters with underlines represent intronsequences. Letters with no underline represent exon sequences. Numbersindicate nucleotide positions of the AAV cap ORF. SD, splice donor; SA,splice acceptor. Please note that SEQ ID NO: 199 of AAV1 and SEQ ID NO:199 of AAV3 are corresponding to each other in sequence alignment.Likewise, SEQ ID NO: 213 of AAV1 and SEQ ID NO: 213 of AAV3 arecorresponding to each other in sequence alignment.

In the course of the experiment, when the AAV9 cap gene ORF wasexpressed in an antisense orientation in HEK293 cells or Neuro2a cells,the majority of the antisense AAV9 cap gene mRNA-derived RT-PCR productswere truncated by approximately 1.7 kb (FIG. 4 ), although this was notthe case with the RNA recovered from theAAV-PHP.B-hSynl-GFP-TRADE-transduced mouse brain tissue (FIG. 3 ).Sequencing of the truncated RT-PCR products revealed that a 1694 bp-longregion was missing within the AAV9 cap ORF (FIG. 5 ). Without beingbound by any particular theory, it appears that the truncation resultsfrom a splicing event, based on the observation that we could identifysplice donor and acceptor sites in the PCR products that have the commonfeatures of exon-intron junctions. Intriguingly, a sequence alignmentstudy revealed that the cryptic splice donor and acceptor sites with thecommon features of exon-intron junctions can also be identified in manynaturally occurring AAV serotypes at the regions corresponding to thesplice donor and acceptor sites identified in the AAV9 cap gene and theyare highly conserved (FIG. 6 ). This indicates that splicing couldpotentially take place in the cap ORF-derived antisense mRNA of not onlyAAV9 but also many other AAV strains. To date, we have found thatsplicing occurs on the AAV3 cap ORF-derived antisense mRNA when it isexpressed under the control of a human liver-specific promoter (LSP) inHepG2 cells. Although full characterization has not yet been completed,a preliminary RT-PCR using an antisense mRNA-specific RT primer yieldedtruncated RT-PCR products in addition to the full-length, non-splicedproduct. The sequencing analysis of two truncated RT-PCR productsrevealed that there were multiple splicing events on the antisense mRNA(FIG. 7 ). A sequencing alignment study has identified additionalpotential splice donor and acceptor sites (FIG. 8 and FIG. 9 ). We alsofound splicing events in the antisense mRNA derived from the AAV1 capORF when antisense mRNA was transcribed by the hSynl enhancer-promoterin HEK293 cells or Neuro2a cells (FIG. 10 ). Many of the identifiedsplice donor sites (GT/CA) and splice acceptor sites (AG/TC) are highlyconserved across different serotypes, indicating the possibility thatthese sites are also utilized as splicing donor and acceptor sites inthe AAV serotypes that have yet to be investigated. Indeed, we havefound that splicing of antisense mRNA transcripts of the AAV1, AAV3 andAAV9 cap ORFs uses several common splice donor and acceptor sites (FIG.10 ). To date, we have not yet observed splicing of antisense mRNAtranscripts of the AAV5 cap ORF. For serotypes other than AAV1, 3, 5 and9, splicing events in antisense mRNA of the AAV cap ORFs have not yetbeen investigated.

Potential splicing of the cap ORF-derived antisense mRNA isscientifically intriguing, but may hinder the TRADE system when thefull-length cap ORF sequence needs to be recovered from antisense mRNA.To overcome this potential issue, we introduced silent mutations thatpresumably disrupt the conserved sequences at exon-intron junctions andbranching points. To demonstrate proof of principle of this approach, weintroduced silent mutations into the AAV9 cap ORF contained in theplasmid, pAAV9-N272A-PHP.B-hSyn1-GFP-TRADE, that disrupt the spliceacceptor (SA) consensus sequence (pAAV9NS1 construct), the splice donor(SD) consensus sequence (pAAV9NS2 construct), and both the spliceacceptor and donor consensus sequences (pAAV9NS3 construct). Please notethat NS stands for “non-spliced.” The method we use to disrupt theseconsensus sequences is described below.

We codon-optimize the AAV cap ORF sequence for human cell expression.

To identify potential splice donor and acceptor sites on antisense mRNAderived from the cap ORFs, we develop and use our proprietary databaseof potential splice donor and acceptor sites on antisense mRNA based onour experimental and bioinformatics observations (i.e., FIGS. 5, 6, 7,8, 9 and 10 ).

We destroy the GT (splice donor) and / or AG (splice acceptor) consensussequence by changing at least one nucleotide using the codon-optimizedsequence. If the codon-optimized sequence is not applicable, we use analternative nucleotide(s) that can destroy the consensus sequence.

We remove a stretch of T’s upstream of the splice acceptor sites byintroducing silence mutations based on the codon-optimized sequence. Ifthe codon-optimized sequence is not sufficient to destroy a stretch ofT’s, we use alternative nucleotides.

We also avoid G at the exon termini as much as possible.

Using several programs that can predict branching points (e.g., HumanSplicing Finder (Desmet, Hamroun et al. 2009)), we identify potentialbranching points and replace them with the codon-optimized sequence. Ifthe degree of nucleotide changes attainable by this method is notsufficient, we introduce alternative nucleotides to disrupt potentialbranching points.

With this method, we have created AAV9NS1 (SA, destroyed), AAV9NS2 (SD,destroyed) and AAV9NS3 (both SD and SA, destroyed) cap ORFs (FIG. 9 ).We expressed these ORFs in an antisense orientation under the control ofthe hSynl enhancer-promoter in Neuro2a cells by transient plasmidtransfection, and analyzed the antisense transcripts by RT-PCR. Thisexperiment revealed that the splicing could be effectively suppressed inall of the NS1, NS2 and NS3 cap ORFs (FIG. 10 ). It should be noted thateven if splicing takes place on the cap ORF-derived antisense mRNA, itwould still be possible to recover the relatively small peptideinsertion region of the cap ORF by RT-PCR from pre-mRNA.

The TRADE method described herein uses antisense mRNA for viral proteinevolution to establish the proof-of-principle and to show successfulreduction of the method to practice. The TRADE system can also utilizemRNA in a sense orientation as long as the viruses can be produced andpotential expression of viral proteins in target cells during thedirected evolution procedure does not hinder successful evolution ofnovel capsids.

Additional information related to nucleic acid splicing and AAV may befound in Desmet et al., Nucleic Acids Res 37, e67 (2009); Matsuzaki etal., Neurosci Lett 665, 182-188 (2018); and Hordeaux et al., Mol Ther26,664-668 (2018).

All references cited in this disclosure are incorporated by reference intheir entirety.

1-19. (canceled)
 20. A nucleic acid comprising: A Parvoviridae genomeflanked by ITR sequences, wherein the Parvoviridae genome comprises aParvoviridae intron, a Parvoviridae cap gene, and a firstpolyadenylation signal in a first orientation; A first promoter in thefirst orientation that drives expression of the Parvoviridae cap gene inthe presence of adenoviral helper functions; and A second promoter inthe first orientation that drives expression of the Parvoviridae capgene in the absence of adenoviral helper functions.
 21. The nucleic acidof claim 20, wherein the second promoter is a cell type-specificpromoter.
 22. The nucleic acid of claim 20, wherein the second promoteris a ubiquitous promoter.
 23. The nucleic acid of claim 20, wherein theParvoviridae cap gene is a wild-type AAV cap gene.
 24. The nucleic acidof claim 23, wherein the AAV cap gene sequences is the AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or othernatural AAV isolate cap gene sequence.
 25. The nucleic acid of claim 20,wherein the Parvoviridae cap gene is an engineered AAV cap gene.
 26. Thenucleic acid of claim 20, wherein the Parvoviridae cap gene is one of alibrary of diverse AAV cap genes.
 27. A nucleic acid library comprisinga plurality of nucleic acids of claim 20, wherein the nucleic acidscomprise a plurality of unique Parvoviridae cap gene sequences.
 28. Thenucleic acid library of claim 27, wherein the nucleic acid librarycomprises greater than about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ uniqueAAV cap gene sequences.
 29. The nucleic acid of claim 20, furthercomprising a gene of interest.
 30. A method for identifying an AAVvector with a cap gene sequence that has increased ability to transducecells from a tissue of interest when compared to at least one other AAVvector with a different cap gene sequence, the method comprising:Preparing a first-round AAV TRADE vector library by introducing thenucleic acid library of claim 27 into an AAV packaging cell line andrecovering the first round AAV TRADE vector library from the packagingcell line; Injecting one or more animals with the first-round AAV TRADEvector library; Recovering cap gene sequences of AAV vectors that areenriched in cells of the tissue of interest in the animals; Preparing asecond-round AAV TRADE nucleic acid library comprising recovered capgene sequences of the enriched AAV vectors and introducing this libraryinto an AAV packaging cell line and recovering the second round AAVTRADE vector library from the packaging cell line; Performing a secondround of enrichment by injecting one or more animals with thesecond-round AAV TRADE vector library and recovering cap gene sequencethat are enriched in cells of the tissue of interest in the animals; andIdentifying enriched AAV cap gene sequences after the first-roundenrichment, after the second-round enrichment, and after any subsequentrounds of enrichment. 31-38. (canceled)
 39. An AAV cap ORF sequencecomprising one or more following mutations in the exon-intron junctionsat splicing donor sites: AAV1 VP1 cap ORF1009-CTTAC(junction)CAGCA-1018* (SEQ ID NO:199) AAV3 VP1 cap ORF1006-CTTAC(junction)CAGCA-1015* (SEQ ID NO:199) AAV1 VP1 cap ORF1228-TTTAC(junction)CTTCA-1237 (SEQ ID NO:200) AAV3 VP1 cap ORF1237-TATAC(junction)CTTCG-1246 (SEQ ID NO:201) AAV1 VP1 cap ORF1331-ATTAC(junction)CTGAA-1340 (SEQ ID NO:202) AAV1 VP1 cap ORF1434-GCTAC(junction)CTGGA-1443 (SEQ ID NO:203) AAV1 VP1 cap ORF1502-TTTAC(junction)CTGGA-1510 (SEQ ID NO:204) AAV1 VP1 cap ORF1803-ATTAC(junction)CTGGC-1812 (SEQ ID NO:205) AAV3 VP1 cap ORF1803-CTTAC(junction)CTGGC-1812 (SEQ ID NO:206) AAV1 VP1 cap ORF1835-TGTAC(junction)CTGCA-1844 (SEQ ID NO:207) AAV1 VP1 cap ORF2189-GTTAC(junction)CTTAC-2198 (SEQ ID NO:208) AAV9 VP1 cap ORF2189-GATAC(junction)CTGAC-2198 (SEQ ID NO:209) AAV1 VP1 cap ORF2194-CTTAC(junction)CCGTC-2203 (SEQ ID NO:210) AAV3 VP1 cap ORF2194-CTCAC(junction)ACGAA-2203 (SEQ ID NO:211).
 40. (canceled) 41.(canceled)